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Sample records for gill cd binding

  1. Interactive effects of waterborne metals in binary mixtures on short-term gill-metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Niyogi, Som; Nadella, Sunita R; Wood, Chris M

    2015-08-01

    Metal binding to fish gills forms the basis of the biotic ligand model (BLM) approach, which has emerged as a useful tool for conducting site-specific water quality assessments for metals. The current BLMs are designed to assess the toxicity of individual metals, and cannot account for the interactive effects of metal mixtures to aquatic organisms including fish. The present study was designed mainly to examine the interactive effects of waterborne metals (Cd, Zn, Cu, Ag, and Ni) in specific binary combinations on short-term (3h) gill-metal binding and essential ion (Ca(2+) and Na(+)) uptake (a physiological index of toxicity) in fish, using juvenile freshwater rainbow trout (Oncorhynchus mykiss) as the model species. We hypothesized that binary mixtures of metals that share a common mode of uptake and toxicity (e.g., Cd and Zn - Ca(2+) antagonists, Cu and Ag - Na(+) antagonists) would reduce the gill binding of each other via competitive interactions and induce less than additive effects on ion transport. In addition, the mixture of metals that have different modes of uptake and toxicity (e.g., Cd and Cu, or Cd and Ni) would not exhibit any interactive effects either on gill-metal binding or ion transport. We found that both Zn and Cu reduced gill-Cd binding and vice versa, however, Ni did not influence gill-Cd binding in fish. Surprisingly, Ag was found to stimulate gill-Cu binding especially at high exposure concentrations, whereas, Cu had no effect on gill-Ag binding. The inhibitory effect of Cd and Zn in mixture on branchial Ca(2+) uptake was significantly greater than that of Cd or Zn alone. Similarly, the inhibitory effect of Cu and Ag in mixture on branchial Na(+) uptake was significantly greater than that of Cu or Ag alone. The inhibitory effects of Cd and Zn mixture on Ca(2+) uptake as well as Cu and Ag mixture on Na(+) uptake were found to follow the principles of simple additivity. In contrast, no significant additive effect on either Ca(2+) or Na

  2. Copper binding affinity of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) gills: Implications for assessing bioavailable metal

    SciTech Connect

    MacRae, R.K.; Smith, D.E.; Swoboda-Colberg, N.; Meyer, J.S.; Bergman, H.L. . Dept. of Zoology and Physiology)

    1999-06-01

    In this study, the authors determined the conditional stability constant (log K[prime]) of copper for the gills of rainbow trout (Oncorhynchus mykiss; RBT) and brook trout (Salvelinus fontinalis; BT). Using toxicity-based complexation bioassays, which measure the effect of competing organic ligands on copper toxicity, the RBT gill copper log K[prime] range was 6.4 to 7.2. Using a Scatchard analysis of gill Cu accumulation, the RBT log K[prime] was 7.50 and the BT log K[prime] was 7.25. The close agreement in RBT log K[prime] values between these two methods suggests that measurement of gill copper accumulation is an acceptable alternative for determining a toxicity-based gill copper binding affinity. The results also suggest that there is either a single gill copper binding component or, more realistically, multiple components with similar binding properties that function collectively to define a single toxicologically relevant copper conditional stability constant. These results suggest analytical approaches to measuring bioavailable metal concentrations, such as geochemical modeling where biological ligands are included in speciation calculations, may adequately simulate complex biological ligands. A method to convert gill copper accumulation to a bioavailable water criterion is also discussed.

  3. Genotoxic Effects Induced by Cd(+2), Cr(+6), Cu(+2) in the Gill and Liver of Odontesthes bonariensis (Piscies, Atherinopsidae).

    PubMed

    Gasulla, J; Picco, S J; Carriquiriborde, P; Dulout, F N; Ronco, A E; de Luca, J C

    2016-05-01

    Genotoxic effects of Cd(+2), Cr(+6), and Cu(+2) on the gill and liver of the Argentinean Silverside (Odontesthes bonariensis) were studied using the comet assay and in relation with the metal tissue accumulation. Fish were exposed to three waterborne concentrations of each metal for 2 and 16 days. Genotoxicity was assessed by the single cell gel electrophoresis (comet assay). After 2 days, significant increase of the genetic damage index (GDI) was only observed in the gill of fish exposed to Cr(+6) and Cu(+2), and the LOECs were 2160 nM and 921.1 nM, respectively. The gill LOEC for Cd(+2) by 16 days was 9.4 nM. In the liver, LOECs were obtained only for Cd(+2) and Cr(+6) and were 9.4 and 2160 nM, respectively. The three metals were able to induce genotoxic effects at environmentally relevant concentrations and the gill was the most sensitive organ.

  4. DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

    PubMed Central

    Varotto, Laura; Domeneghetti, Stefania; Rosani, Umberto; Manfrin, Chiara; Cajaraville, Miren P.; Raccanelli, Stefano; Pallavicini, Alberto; Venier, Paola

    2013-01-01

    Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs. PMID:23355883

  5. Toxicity and Traces of Hg, Pb and Cd in the Hepatopancreas, Gills and Muscles of Perna viridis from Jakarta Bay, Indonesia.

    PubMed

    Irnidayanti, Y

    2015-02-01

    Heavy metals contamination on the coast of Jakarta Bay has led to the level of pollution and can cause toxicity to organisms living in the sea, i.e., green mussels. Green mussels have the ability to detoxify metals entering their bodies. Their ability to accumulate metals is higher than other aquatic animals. This is due to their sedentary life which prevents them from avoiding the effects of pollution and their high tolerance to certain metals. The high concentration of metal content would be toxic to the cell because metal ions can act as oxidants and bind to organic and protein molecules. The results of the study showed that traces of heavy metals were detected in the hepatopancreas, gills, muscles and gonads organs of the mussels living in the waters of Muara Angke. Lead (Pb) and cadmium (Cd) were found in all four organs, while mercury (Hg) was not detected in the muscles. Traces of Hg and Cd were not detected in hepatopancreas, gills, muscles and gonads of green mussels in Panimbang, while Pb was detected by 0.00 1 in the male gonads and 0.01 in hepatopancreas. The concentration of Pb in the male gonads are still below the acceptable limit and concentration of Pb in the hepatopancreas is relatively equivalent to the acceptable limit. Metal detection in the organs above shows that the Muara Angke waters tend to be polluted and have an impact on the mussels weight loss as a result of heavy metal toxicity.

  6. Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd.

    PubMed

    Vincent-Hubert, Françoise; Arini, Adeline; Gourlay-Francé, Catherine

    2011-07-14

    The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.

  7. Influence of natural organic matter source on copper speciation as demonstrated by Cu binding to fish gills, by ion selective electrode, and by DGT gel sampler

    USGS Publications Warehouse

    Luider, C.D.; Crusius, J.; Playle, R.C.; Curtis, P.J.

    2004-01-01

    Rainbow trout (Oncorhynchus mykiss, 2 g) were exposed to 0-5 ??M total copper in ion-poor water for 3 h in the presence or absence of 10 mg C/L of qualitatively different natural organic matter (NOM) derived from water spanning a large gradient in hydrologic residence time. Accumulation of Cu by trout gills was compared to Cu speciation determined by ion selective electrode (ISE) and by diffusive gradients in thin films (DGT) gel sampler technology. The presence of NOM decreased Cu uptake by trout gills as well as Cu concentrations determined by ISE and DGT. Furthermore, the source of NOM influenced Cu binding by trout gills with high-color, allochthonous NOM decreasing Cu accumulation by the gills more than low-color autochthonous NOM. The pattern of Cu binding to the NOM measured by Cu ISE and by Cu accumulation by DGT samplers was similar to the fish gill results. A simple Cu-gill binding model required an NOM Cu-binding factor (F) that depended on NOM quality to account for observed Cu accumulation by trout gills; values of Fvaried by a factor of 2. Thus, NOM metal-binding quality, as well as NOM quantity, are both important when assessing the bioavailability of metals such as Cu to aquatic organisms.

  8. Rabbit CD200R binds host CD200 but not CD200-like proteins from poxviruses

    PubMed Central

    Akkaya, Munir; Kwong, Lai-Shan; Akkaya, Erdem; Hatherley, Deborah; Barclay, A. Neil

    2016-01-01

    CD200 is a widely distributed membrane protein that gives inhibitory signals through its receptor (CD200R) on myeloid cells. CD200 has been acquired by herpesviruses where it has been shown to interact with host CD200R and downmodulate the immune system. It has been hypothesized that poxviruses have acquired CD200; but the potential orthologues show less similarity to their hosts. Myxoma virus M141 protein is a potential CD200 orthologue with a potent immune modulatory function in rabbits. Here, we characterized the rabbit CD200, CD200R and tested the CD200-like sequences for binding CD200R. No binding could be detected using soluble recombinant proteins, full length protein expressed on cells or myxoma virus infected cells. Finally, using knockdown models, we showed that the inhibitory effect of M141 on RAW 264.7 cells upon myxoma virus infection is not due to CD200R. We conclude that the rabbit poxvirus CD200-like proteins cause immunomodulation without utilizing CD200R. PMID:26590792

  9. Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis.

    PubMed

    Katsumiti, A; Gilliland, D; Arostegui, I; Cajaraville, M P

    2014-08-01

    CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5 nm) at a wide range of concentrations (0.001-100 mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31-5 mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on phagocytosis were found in hemocytes exposed to bulk CdS and to CdS QDs at concentrations equal or higher than 1.25 mg Cd/L but not in those exposed to ionic Cd, indicating a particle-specific effect on phagocytosis. In conclusion, cell-mediated immunity and gill cell function represent significant targets for CdS QDs toxicity.

  10. Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

    NASA Astrophysics Data System (ADS)

    Pan, Luqing; Liu, Na; Zhang, Hongxia; Wang, Jing; Miao, Jingjing

    2011-06-01

    There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 μg.L-1), Pb2+ (50, 250, and 500 μg L-1) and Cd2+ (5, 25, and 50 μg L-1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage ( F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 μg L-1 Pb2+ treatment group at day 3 and the 50 μg L-1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 μg L-1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.

  11. CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

    PubMed

    Jay, Anthony G; Chen, Alexander N; Paz, Miguel A; Hung, Justin P; Hamilton, James A

    2015-02-20

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

  12. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    SciTech Connect

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  13. Differential peptide binding to CD40 evokes counteractive responses.

    PubMed

    Khan, Srijit; Alonso-Sarduy, Livan; Alonso, Livan; Roduit, Charles; Bandyopadhyay, Syamdas; Singh, Shailza; Saha, Shipra; Tacchini-Cottier, Fabienne; Roy, Somenath; Dietler, Giovanni; Kasas, Sandor; Das, Pradeep; Krishnasastry, M V; Saha, Bhaskar

    2012-05-01

    The antigen-presenting cell–expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)–12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

  14. Binding of nickel and copper to fish gills predicts toxicity when water hardness varies, but free-ion activity does not

    SciTech Connect

    Meyer, J.S.; Bobbitt, J.P.; Debrey, L.D.; Boese, C.J.; Bergman, H.L.; Santore, R.C.; Paquin, P.R.; Ditoro, D.M.; Allen, H.E.

    1999-03-15

    Based on a biotic-ligand model (BLM), the authors hypothesized that the concentration of a transition metal bound to fish gills ([M{sub gill}]) will be a constant predictor of mortality, whereas a free-ion activity model is generally interpreted to imply that the chemical activity of the aquo (free) ion of the metal will be a constant predictor of mortality. In laboratory tests, measured [Ni{sub gill}] and calculated [Cu{sub gill}] were constant predictors of acute toxicity of Ni and Cu to fathead minnows (Pimephales promelas) when water hardness varied up to 10-fold, whereas total aqueous concentrations and free-ion activities of Ni and Cu were not. Thus, the BLM, which simultaneously accounts for (a) metal speciation in the exposure water and (b) competitive binding of transition-metal ions and other cations to biotic ligands predicts acute toxicity better than does free-ion activity of Ni or Cu. Adopting a biotic-ligand modeling approach could help establish a more defensible, mechanistic basis for regulating aqueous discharges of metals.

  15. CD4-Induced Antibodies Promote Association of the HIV-1 Envelope Glycoprotein with CD4-Binding Site Antibodies

    PubMed Central

    Fellinger, Christoph H.; Prasad, Neha R.; Zhou, Amber S.; Kondur, Hema R.; Joshi, Vinita R.; Quinlan, Brian D.; Farzan, Michael

    2016-01-01

    ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5. IMPORTANCE Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that

  16. Measurement of p-nitrophenyl acetate esterase activity (EA), total antioxidant capacity (TAC), total oxidant status (TOS) and acetylcholinesterase (AChE) in gills and digestive gland of Mytilus galloprovincialis exposed to binary mixtures of Pb, Cd and Cu.

    PubMed

    Franco-Martinez, Lorena; Romero, Diego; García-Navarro, José A; Tecles, Fernando; Teles, Mariana; Tvarijonaviciute, Asta

    2016-12-01

    The aims of the present work were (1) to evaluate oxidative stress biomarkers and AChE in two tissues of wild mussel (Mytilus galloprovincialis) of high biochemical activity and accumulation capacity (gills and digestive gland) and (2) to study the behaviour of these biomarkers in presence of heavy metals. For this, EA, TOS, TAC and AChE were measured in tissues of mussels exposed to binary combination of Pb, Cd and Cu. Mussels (n = 36) were exposed to one of the binary mixtures of Pb (1000 μg L(-1)), Cd (100 μg L(-1)) and Cu (100 μg L(-1)) for 7 days, under controlled conditions. Gills and digestive gland were extracted and frozen at -80 °C until analysis. The automatic methods employed for the measurement of EA, TAC, TOS and AChE in M. galloprovincialis revealed higher levels of these biomarkers in digestive gland than gills. Study results suggest that gills would be the tissue of election for study oxidative stress markers, whereas digestive tissue should be selected for AChE measurements in case of evaluation of combined metal toxicity in mussels.

  17. CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo.

    PubMed

    Haas, Karen M; Sen, Suman; Sanford, Isaac G; Miller, Ann S; Poe, Jonathan C; Tedder, Thomas F

    2006-09-01

    The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.

  18. CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

    PubMed

    Ni, Xiaoling

    2012-03-01

    People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system.

  19. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    SciTech Connect

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-02-20

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  20. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6.

    PubMed

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H; Orian-Rousseau, Véronique

    2015-06-29

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.

  1. Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids.

    PubMed Central

    Yu, B; Hailman, E; Wright, S D

    1997-01-01

    Lipopolysaccharide binding protein (LBP) is a plasma protein known to facilitate the diffusion of bacterial LPS (endotoxin). LBP catalyzes movement of LPS monomers from LPS aggregates to HDL particles, to phospholipid bilayers, and to a binding site on a second plasma protein, soluble CD14 (sCD14). sCD14 can hasten transfer by receiving an LPS monomer from an LPS aggregate, and then surrendering it to an HDL particle, thus acting as a soluble "shuttle" for an insoluble lipid. Here we show that LBP and sCD14 shuttle not only LPS, but also phospholipids. Phosphatidylinositol (PI), phosphatidylcholine, and a fluorescently labeled derivative of phosphatidylethanolamine (R-PE) are each transferred by LBP from membranes to HDL particles. The transfer could be observed using recombinant LBP and sCD14 or whole human plasma, and the plasma-mediated transfer of PI could be blocked by anti-LBP and partially inhibited by anti-CD14. sCD14 appears to act as a soluble shuttle for phospholipids since direct binding of PI and R-PE to sCD14 was observed and because addition of sCD14 accelerated transfer of these lipids. These studies define a new function for LBP and sCD14 and describe a novel mechanism for the transfer of phospholipids in blood. In further studies, we show evidence suggesting that LBP transfers LPS and phospholipids by reciprocal exchange: LBP-catalyzed binding of R-PE to LPS x sCD14 complexes was accompanied by the exit of LPS from sCD14, and LBP-catalyzed binding of R-PE to sCD14 was accelerated by prior binding of LPS to sCD14. Binding of one lipid is thus functionally coupled with the release of a second. These results suggest that LBP acts as a lipid exchange protein. PMID:9006000

  2. High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

    PubMed Central

    Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

    2010-01-01

    Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

  3. Gilles de la Tourette syndrome and disruptive behavior disorders: prevalence, associations, and explanation of the relationships.

    PubMed

    Robertson, Mary M; Cavanna, Andrea E; Eapen, Valsamma

    2015-01-01

    Gilles de la Tourette syndrome and conduct disorder (CD) are both heterogeneous childhood onset conditions, and although patients with CD have been described in Gilles de la Tourette syndrome cohorts, little is known about the etiology of CD in Gilles de la Tourette syndrome or of the interrelationships. A cohort of 578 consecutive patients with Gilles de la Tourette syndrome was assessed using standard assessment protocols. A total of 13.5% of participants had only Gilles de la Tourette syndrome, whereas the rest had associated comorbidities and psychopathology. CD occurred in 14.5% of Gilles de la Tourette syndrome probands. These findings suggest that CD is not an integral part of Gilles de la Tourette syndrome but rather that CD in the context of Gilles de la Tourette syndrome is related to the presence of attention deficit hyperactivity disorder, as well as, and importantly, a family history of aggressive and violent behavior and forensic encounters.

  4. Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes

    PubMed Central

    Horvatinovich, Joe M.; Grogan, Elizabeth W.; Norris, Marcus; Steinkasserer, Alexander; Lemos, Henrique; Mellor, Andrew L.; Tcherepanova, Irina Y.; Nicolette, Charles A.

    2017-01-01

    The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can attenuate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R–associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2–dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unresponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell unresponsiveness. PMID:28193829

  5. Antibody raised against soluble CD4-rgp120 complex recognizes the CD4 moiety and blocks membrane fusion without inhibiting CD4-gp120 binding

    PubMed Central

    1990-01-01

    We studied the humoral response of mice immunized with soluble CD4- rgp120 complex, testing polyclonal and monoclonal antibodies (mAbs) with the aim of identifying molecular changes that take place after the first interaction between human immunodeficiency virus and the cell surface. The antisera had a paradoxically high syncytia-blocking titer associated with anti-CD4 specificity, while their capacity to inhibit CD4-gp120 binding was relatively modest. One of the mAbs produced from these responders blocks syncytia formation but does not inhibit CD4 interaction with gp120. Apparently, this mAb interacts with the CD4 moiety of CD4-gp120 complex and prevents a post-binding event necessary for membrane fusion and viral infection. PMID:2212945

  6. Influence of surfactants on gill physiology and cadmium uptake in perfused rainbow trout gills

    SciTech Connect

    Paert, P.S.; Svanberg, O.; Bergstroem, E.

    1985-04-01

    Cadmium transfer through and the retention of metal in perfused gills from rainbow trout (Salmo gairdneri) has been studied in the presence of two detergents, LAS (linear alkylaryl sulphonate) and NP-10EO (nonylphenol ethoxylate). Accordingly, the effects of the metal and the surfactants on gill viability (vascular resistance, oxygen diffusion capacity, sodium net flux) was measured. Cd had no effect on gill viability either at 0.008 or at 9.0 mumol/liter during a 60-min perfusion period. The viability of the gills deteriorated markedly during 60 min of exposure to 100 mumol/liter LAS and to NP-10EO, or to a mixture of 100 mumol/liter surfactant + 8.1-8.3 mumol/liter Cd. LAS, 100 mumol/liter, reduced Cd transfer, whereas NP-10EO had no effect. NP-10EO increased Cd retention in gill tissue. LAS more than doubled Cd transfer through the gills when tested in concentrations expected to be found in a polluted recipient (0.9 micrograms/liter Cd + 0.05 mg/liter LAS). NP-10EO had no effect on the transfer when tested under these environmentally relevant conditions.

  7. Comparative study of the bioaccumulation and elimination of trace metals (Cd, Pb, Zn, Mn and Fe) in the digestive gland, gills and muscle of bivalve Pinna nobilis during a field transplant experiment.

    PubMed

    Jebali, Jamel; Chouba, Lassaad; Banni, Mohamed; Boussetta, Hamadi

    2014-04-01

    The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.

  8. Critical and direct involvement of the CD23 stalk region in IgE binding

    PubMed Central

    Selb, Regina; Eckl-Dorna, Julia; Twaroch, Teresa E.; Lupinek, Christian; Teufelberger, Andrea; Hofer, Gerhard; Focke-Tejkl, Margarete; Gepp, Barbara; Linhart, Birgit; Breiteneder, Heimo; Ellinger, Adolf; Keller, Walter; Roux, Kenneth H.; Valenta, Rudolf; Niederberger, Verena

    2017-01-01

    Background The low-affinity receptor for IgE, FcεRII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of transepithelial allergen migration. Objective We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. Methods We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N-linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. Results A hitherto unknown IgE-binding site was mapped on the stalk region of CD23, and the non–N-glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti-IgE antibody, omalizumab, which inhibits IgE binding to FcεRI, also inhibited IgE binding to CD23. Conclusion Our results provide a new model for the CD23-IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti-IgE antibody omalizumab. PMID:27343203

  9. Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

    SciTech Connect

    Ruffell, Brian; Johnson, Pauline . E-mail: pauline@interchange.ubc.ca

    2005-08-26

    CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.

  10. CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

    PubMed Central

    Kellett-Clarke, Helena; Stegmann, Monika; Barclay, A. Neil; Metcalfe, Clive

    2015-01-01

    CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies. PMID:26379032

  11. Identification of ligands that target the HCV-E2 binding site on CD81

    NASA Astrophysics Data System (ADS)

    Olaby, Reem Al; Azzazy, Hassan M.; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  12. Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

    PubMed

    Goswami, Debashree; März, Sigrid; Li, Yu-Tung; Artz, Annette; Schäfer, Kerstin; Seelige, Ruth; Pacheco-Blanco, Mariana; Jing, Ding; Bixel, Maria Gabriele; Araki, Masatake; Araki, Kimi; Yamamura, Ken-Ichi; Vestweber, Dietmar

    2017-03-30

    CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

  13. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

    PubMed Central

    Yang, B; Yang, B L; Savani, R C; Turley, E A

    1994-01-01

    We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses

  14. CD44v6-Peptide Functionalized Nanoparticles Selectively Bind to Metastatic Cancer Cells.

    PubMed

    Li, Linxian; Schmitt, Mark; Matzke-Ogi, Alexandra; Wadhwani, Parvesh; Orian-Rousseau, Veronique; Levkin, Pavel A

    2017-01-01

    CD44v6 peptide functionalized nanoparticles are fabricated in a facile and controllable way to selectively bind to CD44v6 positive tumor cells with highly efficient anticancer and antimetastatic properties. The reported modular synthesis and facile preparation makes this system highly potent for developing novel multifunctional nanocarriers for therapeutic and/or diagnostic anticancer applications.

  15. Gills of antarctic fish.

    PubMed

    Rankin, J C; Tuurala, H

    1998-01-01

    We review the literature on the way the structure of icefish gills relates the physiology of these haemoglobin-less fishes. Vascular casting confirmed earlier reports that the only special feature of the gills is the large size of the blood vessels, especially the prominent and continuous marginal channels Isolated perfused gill arches were used to study the effects of changes in afferent and efferent pressure on gill resistance and tritiated water influx in Chionobathyscus dewitti. Increasing perfusion rate did not change gill resistance, but there were moderate proportional increases in water influx. Reducing efferent pressure increased gill resistance but did not affect water influx. In both C. dewitti and Cryodraco antarcticus gills perfused at constant flow rate, noradrenaline produced concentration-dependent decreases in gill resistance and, with high concentrations, increases in water influx. Fixation while perfusion continued was used to compare blood space dimensions in control, noradrenaline-treated and unperfused gills. Noradrenaline caused large increases in the thickness of the lamellar blood space and increased lamellar height, despite a greatly reduced afferent pressure. This suggests that modulation of pillar cell active tension might be involved in control of lamellar perfusion. The possible relationship between gill water fluxes and lamellar recruitment is discussed.

  16. Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation.

    PubMed

    Shiba, Emiko; Izawa, Kumi; Kaitani, Ayako; Isobe, Masamichi; Maehara, Akie; Uchida, Koichiro; Maeda, Keiko; Nakano, Nobuhiro; Ogawa, Hideoki; Okumura, Ko; Kitamura, Toshio; Shimizu, Toshiaki; Kitaura, Jiro

    2017-02-17

    LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f(-/-) mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f(-/-) mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo.

  17. Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation*

    PubMed Central

    Shiba, Emiko; Izawa, Kumi; Kaitani, Ayako; Isobe, Masamichi; Maehara, Akie; Uchida, Koichiro; Maeda, Keiko; Nakano, Nobuhiro; Ogawa, Hideoki; Okumura, Ko; Kitamura, Toshio; Shimizu, Toshiaki; Kitaura, Jiro

    2017-01-01

    LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f−/− mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f−/− mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo. PMID:28073916

  18. Characterization of the high molecular weight Cd-binding complex in water hyacinth (Eichhornia crassipes) when exposed to Cd.

    PubMed

    Wu, Jiann-Shing; Ho, Tzu-Chieh; Chien, Hung-Chi; Wu, Yu-Jen; Lin, Shyh-Mirn; Juang, Rong-Huay

    2008-07-23

    Water hyacinth ( Eichhornia crassipes) is a rapid-growing freshwater vascular plant that has been used to remove heavy metals in contaminated water. But the transportation and distribution of the absorbed heavy metal in the plant are not clear. In this study, water hyacinth was exposed to cadmium (Cd, 10 microM, pulse) and then transferred to a Cd-free solution (chase). The Cd content in the tissues was measured, and the Cd-binding complexes were isolated and identified. We found that (1) in two days, up to 80% of the Cd in the solution was absorbed by the plant, and the Cd could not be released back to the growth solution in the chase period; (2) approximately 1 mg of Cd was accumulated in the water hyacinth/g of dry weight in this condition; (3) invading Cd was bound rapidly as the low-molecular-weight (LMW) complex serving as the transient form for further sequestration; (4) the LMW complex in water hyacinth contained no phytochelatins and was different from the LMW complex in fission yeast; (5) the Cd absorbed in the plant was essentially stored in the high-molecular-weight (HMW) form after 1 week; (6) a small fraction of the absorbed Cd was found in the upper part of the plant (stem and leaves) in the form of complexes; (7) the HMW complex was composed of phytochelatins PC 3 and PC 4 primarily, with only a small amount of PC 2; (8) a rare PC-related peptide was found in the HMW complex that might be derived from PC 3. These observations contribute to the further understanding of water hyacinth in serving as an efficient and reliable accumulator for heavy metals.

  19. Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor.

    PubMed

    Kim, Sungwon; Cox, Chasity M; Jenkins, Mark C; Fetterer, Ray H; Miska, Katarzyna B; Dalloul, Rami A

    2014-12-01

    Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surface receptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surface receptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.

  20. Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-Å resolution

    PubMed Central

    Ikemizu, Shinji; Sparks, Lisa M.; van der Merwe, P. Anton; Harlos, Karl; Stuart, David I.; Jones, E. Yvonne; Davis, Simon J.

    1999-01-01

    The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell–cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-Å resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC′C′′ β-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell–cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein–protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies. PMID:10200255

  1. Resistance of primary isolates of human immunodeficiency virus type 1 to soluble CD4 is independent of CD4-rgp120 binding affinity.

    PubMed Central

    Ashkenazi, A; Smith, D H; Marsters, S A; Riddle, L; Gregory, T J; Ho, D D; Capon, D J

    1991-01-01

    The infection of human cells by laboratory strains of human immunodeficiency virus type 1 (HIV-1) can be blocked readily in vitro by recombinant soluble CD4 and CD4-immunoglobulin hybrid molecules. In contrast, infection by primary isolates of HIV-1 is much less sensitive to blocking in vitro by soluble CD4-based molecules. To investigate the molecular basis for this difference between HIV-1 strains, we isolated the gp120-encoding genes from several CD4-resistant and CD4-sensitive HIV-1 strains and characterized the CD4-binding properties of their recombinant gp120 (rgp120) products. Extensive amino acid sequence variation was found between the gp120 genes of CD4-resistant and CD4-sensitive HIV-1 isolates. However, the CD4-binding affinities of rgp120 from strains with markedly different CD4 sensitivities were essentially the same, and only small differences were observed in the kinetics of CD4 binding. These results suggest that the lower sensitivity of primary HIV-1 isolates to neutralization by CD4-based molecules is not due to lower binding affinity between soluble CD4 and free gp120. PMID:1871120

  2. In Vivo Binding and Retention of CD4-Specific DARPin 57.2 in Macaques

    PubMed Central

    Pugach, Pavel; Krarup, Anders; Gettie, Agegnehu; Kuroda, Marcelo; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Trkola, Alexandra; Robbiani, Melissa

    2010-01-01

    Background The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost. Methodology/Principal Findings To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4+ cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4+ cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4+ cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4+ cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation. Conclusions/Significance We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology. PMID:20805996

  3. Increased flexibility and liposome-binding capacity of CD1e at endosomal pH.

    PubMed

    Bushmarina, Natalia; Tourne, Sylvie; Giacometti, Gaëlle; Signorino-Gelo, François; Garcia-Alles, Luis F; Cazenave, Jean-Pierre; Hanau, Daniel; de la Salle, Henri

    2011-06-01

    The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.

  4. Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

    PubMed Central

    Moore, J P; McKeating, J A; Huang, Y X; Ashkenazi, A; Ho, D D

    1992-01-01

    Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins. PMID:1727487

  5. Envelope Conformational Changes Induced by Human Immunodeficiency Virus Type 1 Attachment Inhibitors Prevent CD4 Binding and Downstream Entry Events

    PubMed Central

    Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

    2006-01-01

    BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes. PMID:16571818

  6. Simulation of the coupling between nucleotide binding and transmembrane domains in the ATP binding cassette transporter BtuCD.

    PubMed

    Sonne, Jacob; Kandt, Christian; Peters, Günther H; Hansen, Flemming Y; Jensen, Morten Ø; Tieleman, D Peter

    2007-04-15

    The nucleotide-induced structural rearrangements in ATP binding cassette (ABC) transporters, leading to substrate translocation, are largely unknown. We have modeled nucleotide binding and release in the vitamin B(12) importer BtuCD using perturbed elastic network calculations and biased molecular dynamics simulations. Both models predict that nucleotide release decreases the tilt between the two transmembrane domains and opens the cytoplasmic gate. Nucleotide binding has the opposite effect. The observed coupling may be relevant for all ABC transporters because of the conservation of nucleotide binding domains and the shared role of ATP in ABC transporters. The rearrangements in the cytoplasmic gate region do not provide enough space for B(12) to diffuse from the transporter pore into the cytoplasm, which could suggest that peristaltic forces are needed to exclude B(12) from the transporter pore.

  7. Identification of Inhibitors of CD36-Amyloid Beta Binding as Potential Agents for Alzheimer's Disease.

    PubMed

    Doens, Deborah; Valiente, Pedro A; Mfuh, Adelphe M; X T Vo, Anh; Tristan, Adilia; Carreño, Lizmar; Quijada, Mario; Nguyen, Vu T; Perry, George; Larionov, Oleg V; Lleonart, Ricardo; Fernández, Patricia L

    2017-02-15

    Neuroinflammation is one of the hallmarks of Alzheimer's disease pathology. Amyloid β has a central role in microglia activation and the subsequent secretion of inflammatory mediators that are associated with neuronal toxicity. The recognition of amyloid β by microglia depends on the expression of several receptors implicated in the clearance of amyloid and in cell activation. CD36 receptor expressed on microglia interacts with fibrils of amyloid inducing the release of proinflammatory cytokines and amyloid internalization. The interruption of the interaction CD36-amyloid β compromises the activation of microglia cells. We have developed and validated a new colorimetric assay to identify potential inhibitors of the binding of amyloid β to CD36. We have found seven molecules, structural analogues of the Trichodermamide family of natural products that interfere with the interaction CD36-amyloid β. By combining molecular docking and dynamics simulations, we suggested the second fatty acids binding site within the large luminal hydrophobic tunnel, present in the extracellular domain of CD36, as the binding pocket of these compounds. Free energy calculations predicted the nonpolar component as the driving force for the binding of these inhibitors. These molecules also inhibited the production of TNF-α, IL-6, and IL-1β by peritoneal macrophages stimulated with fibrils of amyloid β. This work serves as a platform for the identification of new potential anti-inflammatory agents for the treatment of Alzheimer's disease.

  8. Visualization of hormone binding proteins in vivo based on Mn-doped CdTe QDs

    NASA Astrophysics Data System (ADS)

    Liu, Fang fei; Yu, Ying; Lin, Bi xia; Hu, Xiao gang; Cao, Yu juan; Wu, Jian zhong

    2014-10-01

    Daminozide (B9) is a growth inhibitor with important regulatory roles in plant growth and development. Locating and quantifying B9-binding proteins in plant tissues will assist in investigating the mechanism behind the signal transduction of B9. In this study, red fluorescent Mn-doped CdTe quantum dots (CdTeMn QDs) were synthesized by a high-temperature hydrothermal process. Since CdTeMn QDs possess a maximum fluorescence emission peak at 610 nm, their fluorescence properties are more stable than those of CdTe QDs. A B9-CdTeMn probe was synthesized by coupling B9 with CdTeMn QDs. The fluorescence intensity of the probe is double that of CdTeMn QDs; its fluorescence stability is also superior under different ambient conditions. The probe retains the biological activity of B9 and is unaffected by interference from the green fluorescent protein present in plants. Therefore, we used this probe to label B9-binding proteins selectively in root tissue sections of mung bean seedlings. These proteins were observed predominantly on the surfaces of the cell membranes of the cortex and epidermal parenchyma.

  9. Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts

    PubMed Central

    1995-01-01

    CD31 is a member of the immunoglobulin superfamily consisting of six Ig- related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM- 1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31- CD31 interactions are critically important only in the initial phases of intercellular adhesion. PMID:7534767

  10. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36.

    PubMed

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127-279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127-279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions.

  11. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36

    PubMed Central

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A.; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127–279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127–279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

  12. Expression of CD3-associated antigen-binding receptors on suppressor T cells.

    PubMed Central

    Kuchroo, V K; Steele, J K; Billings, P R; Selvaraj, P; Dorf, M E

    1988-01-01

    Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules. Images PMID:2973609

  13. IgE binds asymmetrically to its B cell receptor CD23

    PubMed Central

    Dhaliwal, Balvinder; Pang, Marie O. Y.; Keeble, Anthony H.; James, Louisa K.; Gould, Hannah J.; McDonnell, James M.; Sutton, Brian J.; Beavil, Andrew J.

    2017-01-01

    The antibody IgE plays a central role in allergic disease mechanisms. Its effector functions are controlled through interactions between the Fc region and two principal cell surface receptors FcεRI and CD23. The interaction with FcεRI is primarily responsible for allergic sensitization and the inflammatory response, while IgE binding to CD23 is involved in the regulation of IgE synthesis and allergen transcytosis. Here we present the crystal structure of a CD23/IgE-Fc complex and conduct isothermal titration calorimetric binding studies. Two lectin-like “head” domains of CD23 bind to IgE-Fc with affinities that differ by more than an order of magnitude, but the crystal structure reveals only one head bound to one of the two identical heavy-chains in the asymmetrically bent IgE-Fc. These results highlight the subtle interplay between receptor binding sites in IgE-Fc and their affinities, the understanding of which may be exploited for therapeutic intervention in allergic disease. PMID:28361904

  14. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    PubMed

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  15. The structural basis for CD36 binding by the malaria parasite

    PubMed Central

    Hsieh, Fu-Lien; Turner, Louise; Bolla, Jani Reddy; Robinson, Carol V.; Lavstsen, Thomas; Higgins, Matthew K.

    2016-01-01

    CD36 is a scavenger receptor involved in fatty acid metabolism, innate immunity and angiogenesis. It interacts with lipoprotein particles and facilitates uptake of long chain fatty acids. It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. This prevents their destruction by splenic clearance and allows increased parasitaemia. Here we describe the structure of CD36 in complex with long chain fatty acids and a CD36-binding PfEMP1 protein domain. A conserved hydrophobic pocket allows the hugely diverse PfEMP1 protein family to bind to a conserved phenylalanine residue at the membrane distal tip of CD36. This phenylalanine is also required for CD36 to interact with lipoprotein particles. By targeting a site on CD36 that is required for its physiological function, PfEMP1 proteins maintain the ability to tether to the endothelium and avoid splenic clearance. PMID:27667267

  16. The crystal structure of avian CD1 reveals a smaller, more primordial antigen-binding pocket compared to mammalian CD1

    PubMed Central

    Zajonc, Dirk M.; Striegl, Harald; Dascher, Christopher C.; Wilson, Ian A.

    2008-01-01

    The molecular details of glycolipid presentation by CD1 antigen-presenting molecules are well studied in mammalian systems. However, little is known about how these non-classical MHC class I (MHCI) molecules diverged from the MHC locus to create a more complex, hydrophobic binding groove that binds lipids rather than peptides. To address this fundamental question, we have determined the crystal structure of an avian CD1 (chCD1–2) that shares common ancestry with mammalian CD1 from ≈310 million years ago. The chCD1–2 antigen-binding site consists of a compact, narrow, central hydrophobic groove or pore rather than the more open, 2-pocket architecture observed in mammalian CD1s. Potential antigens then would be restricted in size to single-chain lipids or glycolipids. An endogenous ligand, possibly palmitic acid, serves to illuminate the mode and mechanism of ligand interaction with chCD1–2. The palmitate alkyl chain is inserted into the relatively shallow hydrophobic pore; its carboxyl group emerges at the receptor surface and is stabilized by electrostatic and hydrogen bond interactions with an arginine residue that is conserved in all known CD1 proteins. In addition, other novel features, such as an A′ loop that interrupts and segments the normally long, continuous α1 helix, are unique to chCD1–2 and contribute to the unusually narrow binding groove, thereby limiting its size. Because birds and mammals share a common ancestor, but the rate of evolution is slower in birds than in mammals, the chCD1–2-binding groove probably represents a more primordial CD1-binding groove. PMID:19004781

  17. Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation.

    PubMed

    Bowles, Sandra Lyn; Jaeger, Christiane; Ferrara, Claudia; Fingeroth, Joyce; Van De Venter, Maryna; Oosthuizen, Vaughan

    2011-01-01

    IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 "stalk" domain, exCD23>sCD23>derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.

  18. The Phosphatidylserine and Phosphatidylethanolamine Receptor CD300a Binds Dengue Virus and Enhances Infection

    PubMed Central

    Carnec, Xavier; Meertens, Laurent; Dejarnac, Ophélie; Perera-Lecoin, Manuel; Hafirassou, Mohamed Lamine; Kitaura, Jiro; Ramdasi, Rasika; Schwartz, Olivier

    2015-01-01

    ABSTRACT Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PtdSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PtdSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PtdEth and PtdSer present on virions and enhance infection. IMPORTANCE Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300a

  19. CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor

    PubMed Central

    1995-01-01

    Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS- modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS- modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS- modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is

  20. CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

    PubMed Central

    Sarrias, Maria-Rosa; Farnós, Montserrat; Mota, Rubén; Sánchez-Barbero, Fernando; Ibáñez, Anna; Gimferrer, Idoia; Vera, Jorge; Fenutría, Rafael; Casals, Cristina; Yélamos, José; Lozano, Francisco

    2007-01-01

    CD6 is a lymphocyte receptor that belongs to the scavenger receptor cysteine-rich superfamily. Because some members of the scavenger receptor cysteine-rich superfamily act as pattern recognition receptors for microbial components, we studied whether CD6 shares this function. We produced a recombinant form of the ectodomain of CD6 (rsCD6), which was indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding properties) from a circulating form of CD6 affinity-purified from human serum. rsCD6 bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of lipoteichoic acid and LPS, respectively. The Kd of the LPS–rsCD6 interaction was 2.69 ± 0.32 × 10−8 M, which is similar to that reported for the LPS–CD14 interaction. Further experiments showed that membrane CD6 also retains the LPS-binding ability, and it results in activation of the MAPK signaling cascade. In vivo experiments demonstrated that i.p. administration of rsCD6 before lethal LPS challenge significantly improved mice survival, and this was concomitant with reduced serum levels of the proinflammatory cytokines TNF-α, IL6, and IL-1β. In conclusion, our results illustrate the unprecedented bacterial binding properties of rsCD6 and support its therapeutic potential for the intervention of septic shock syndrome or other inflammatory diseases of infectious origin. PMID:17601777

  1. Putative alpha-helical structures in the human immunodeficiency virus type 1 Vpu protein and CD4 are involved in binding and degradation of the CD4 molecule.

    PubMed Central

    Tiganos, E; Yao, X J; Friborg, J; Daniel, N; Cohen, E A

    1997-01-01

    The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a 16-kDa class I integral membrane phosphoprotein with an N-terminal membrane-spanning region and a C-terminal cytoplasmic domain. In the cytoplasmic domain, two amphipathic alpha-helices joined by a flexible turn containing two phosphoacceptor sites have been predicted. Previous studies have shown that Vpu downregulates CD4 molecules by inducing their specific degradation in the endoplasmic reticulum. Phosphorylation of serine residues 52 and 56, present within the cytoplasmic domain of the Vpu protein, has been shown to be essential to this Vpu function. However, the contribution of these two phosphoacceptor sites in the mechanism of CD4 degradation remains undefined. Interestingly, a specific interaction between Vpu and CD4 was recently demonstrated in coimmunoprecipitation experiments. Binding of Vpu was shown to be necessary but not sufficient to mediate CD4 degradation, indicating that interaction between Vpu and CD4 represents an early step critical in triggering a process leading to CD4 degradation. To delineate the sequence(s) and/or structural determinant(s) involved in this Vpu-CD4 interaction and in the Vpu-mediated CD4 degradation, we performed a mutational analysis of the cytoplasmic domain of CD4 and Vpu. Coimmunoprecipitation experiments reveal that disruption of the putative alpha-helical structure in the membrane-proximal cytoplasmic domain of CD4 affects the binding to Vpu, suggesting that this structure may act as an interface for the CD4-Vpu interaction that mediates CD4 degradation. Vpu proteins containing mutations in either or both of the phosphoacceptor sites (Ser52 or/and Ser56) were inactive in regard to CD4 degradation yet retained the capacity to interact with the cytoplasmic domain of CD4. In an attempt to define the minimal region responsible for this interaction, we tested a panel of mutations which were designed to affect the integrity of the putative alpha

  2. Gill's 'History' restored

    NASA Astrophysics Data System (ADS)

    Hurn, Mark

    2009-06-01

    Note about the restoration of the copy of Sir David Gill's 'A History and Description of the Royal Observatory, Cape of Good Hope' in the Library of the Institute of Astronomy, Cambridge. The book was restored with funds provided by the SHA in thanks for facilities for meetings provided to the Institute.

  3. Structural adaptations for ram ventilation: gill fusions in scombrids and billfishes.

    PubMed

    Wegner, Nicholas C; Sepulveda, Chugey A; Aalbers, Scott A; Graham, Jeffrey B

    2013-01-01

    For ram-gill ventilators such as tunas and mackerels (family Scombridae) and billfishes (families Istiophoridae, Xiphiidae), fusions binding the gill lamellae and filaments prevent gill deformation by a fast and continuous ventilatory stream. This study examines the gills from 28 scombrid and seven billfish species in order to determine how factors such as body size, swimming speed, and the degree of dependence upon ram ventilation influence the site of occurrence and type of fusions. In the family Scombridae there is a progressive increase in the reliance on ram ventilation that correlates with the elaboration of gill fusions. This ranges from mackerels (tribe Scombrini), which only utilize ram ventilation at fast cruising speeds and lack gill fusions, to tunas (tribe Thunnini) of the genus Thunnus, which are obligate ram ventilators and have two distinct fusion types (one binding the gill lamellae and a second connecting the gill filaments). The billfishes appear to have independently evolved gill fusions that rival those of tunas in terms of structural complexity. Examination of a wide range of body sizes for some scombrids and billfishes shows that gill fusions begin to develop at lengths as small as 2.0 cm fork length. In addition to securing the spatial configuration of the gill sieve, gill fusions also appear to increase branchial resistance to slow the high-speed current produced by ram ventilation to distribute flow evenly and optimally to the respiratory exchange surfaces.

  4. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    NASA Astrophysics Data System (ADS)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-10-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

  5. Modulating DNA methylation in activated CD8+ T cells inhibits regulatory T cell-induced binding of Foxp3 to the CD8+ T Cell IL-2 promoter.

    PubMed

    Miller, Michelle M; Akaronu, Nnenna; Thompson, Elizabeth M; Hood, Sylvia F; Fogle, Jonathan E

    2015-02-01

    We have previously demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8(+) CTL function in a TGF-β-dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8(+) target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8(+) T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8(+) T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8(+) T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8(+) T cell IL-2 transcription, maturation, and antiviral function.

  6. The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25-CD4+ precursors.

    PubMed

    Skapenko, Alla; Kalden, Joachim R; Lipsky, Peter E; Schulze-Koops, Hendrik

    2005-11-01

    The mechanisms underlying the extrathymic generation of CD25+CD4 regulatory T cells (Tregs) are largely unknown. In this study the IL-4R alpha-chain-binding cytokines, IL-4 and IL-13, were identified as inducers of CD25+ Tregs from peripheral CD25-CD4 naive T cells. IL-4-induced CD25+ Tregs phenotypically and functionally resemble naturally occurring Tregs in that they are anergic to mitogenic stimulation, inhibit the proliferation of autologous responder T cells, express high levels of the Forkhead box P3 and the surface receptors glucocorticoid-induced TNFR family-related protein and CTLA-4, and inhibit effector T cells in a contact-dependent, but cytokine-independent, manner. The IL-4-induced generation of peripheral Tregs was independent of the presence of TGF-beta or IL-10, but was dependent on Ag-specific stimulation and B7 costimulation. The significance of the IL-4Ralpha-binding cytokines in the generation of Ag-specific Tregs was emphasized in a mouse model of oral tolerance, in which neutralization of IL-4 and IL-13 in mice transgenic for the TCR specific for OVA completely inhibited the expansion of OVA-specific Tregs that can be induced in untreated mice by feeding the nominal Ag. Together, our results demonstrate that IL-4 and IL-13 play an important role in generating Forkhead box P3-expressing CD25+ Tregs extrathymically in an Ag-dependent manner and therefore provide an intriguing link between the well-established immunoregulatory capacity of Th2 cells and the powerful CD25+ Treg population. Moreover, our findings might provide the basis for the design of novel therapeutic approaches for targeted immunotherapy with Tregs to known Ags in autoimmune diseases or graft-vs-host reactions.

  7. CD5-CK2 Binding/Activation Deficient Mice Are Resistant to EAE

    PubMed Central

    Axtell, Robert C.; Xu, Liang; Barnum, Scott R.; Raman, Chander

    2009-01-01

    Regulating the differentiation and persistence of encephalitogenic T-cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We recently reported that CD5 has an engagement-dependent prosurvival activity in T-cells which had a direct role in the induction and progression EAE. We predicted that CD5 regulates T-cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector TH-cells that co-express γIFN and IL-17 and to a lesser extent cells that express γIFN or IL-17 in draining lymph nodes and spinal cords. We further show that T-cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation, however following restimulation, they rapidly develop non-responsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T-cell activation and persistence of effector T-cells in neuroinflammatory disease. This study predicts that targeting of γIFN+/IL-17+ TH cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases. PMID:17142752

  8. A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection

    PubMed Central

    1993-01-01

    CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation. PMID:8459222

  9. An enriched stable-isotope approach to determine the gill-zinc binding properties of juvenile rainbow trout (Oncorhynchus mykiss) during acute zinc exposures in hard and soft waters

    USGS Publications Warehouse

    Todd, A.S.; Brinkman, S.; Wolf, R.E.; Lamothe, P.J.; Smith, K.S.; Ranville, J.F.

    2009-01-01

    The objective of the present study was to employ an enriched stable-isotope approach to characterize Zn uptake in the gills of rainbow trout (Oncorhynchus mykiss) during acute Zn exposures in hard water (???140 mg/L as CaCO 3) and soft water (???30 mg/L as CaCO3). Juvenile rainbow trout were acclimated to the test hardnesses and then exposed for up to 72 h in static exposures to a range of Zn concentrations in hard water (0-1,000 ??g/L) and soft water (0-250 ??g/L). To facilitate detection of new gill Zn from endogenous gill Zn, the exposure media was significantly enriched with 67Zn stable isotope (89.60% vs 4.1% natural abundance). Additionally, acute Zn toxicity thresholds (96-h median lethal concentration [LC50]) were determined experimentally through traditional, flow-through toxicity tests in hard water (580 ??g/L) and soft water (110 ??g/L). Following short-term (???3 h) exposures, significant differences in gill accumulation of Zn between hard and soft water treatments were observed at the three common concentrations (75, 150, and 250 ??g/L), with soft water gills accumulating more Zn than hard water gills. Short-term gill Zn accumulation at hard and soft water LC50s (45-min median lethal accumulation) was similar (0.27 and 0.20 ??g/g wet wt, respectively). Finally, comparison of experimental gill Zn accumulation, with accumulation predicted by the biotic ligand model, demonstrated that model output reflected short-term (<1 h) experimental gill Zn accumulation and predicted observed differences in accumulation between hard and soft water rainbow trout gills. Our results indicate that measurable differences exist in short-term gill Zn accumulation following acclimation and exposure in different water hardnesses and that short-term Zn accumulation appears to be predictive of Zn acute toxicity thresholds (96-h LC50s). ?? 2009 SETAC.

  10. The Crystal Structure of CD8alpha,Beta in Complex With YTS156.7.7 Fab And Interaction With Other CD8 Antibodies Define the Binding Mode of CD8alpha,Beta to MHC Class I

    SciTech Connect

    Shore, D.A.; Issafras, H.; Landais, E.; Teyton, L.; Wilson, I.A.

    2009-05-27

    The CD8{alpha}{beta} heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8{alpha} or CD8{beta} to probe the mechanism of CD8{alpha}{beta} binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8{alpha}{beta} immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 {angstrom} resolution. The YTS156.7 epitope on CD8{beta} was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8{beta} are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8{alpha}{beta} interaction, binding of class I tetramers to CD8{alpha}{beta} on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8{alpha}, does not inhibit binding of CD8{alpha}{beta} to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8{alpha}{beta} complex. Together, these data indicate a model for the pMHCI/CD8{alpha}{beta} interaction similar to that observed for CD8{alpha}{alpha} in the CD8{alpha}{alpha}/pMHCI complex, but in which CD8{alpha} occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8{beta} occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8{alpha}{beta} in T cell activation is discussed.

  11. Determination of the Exciton Binding Energy in CdSe Quantum Dots

    SciTech Connect

    Meulenberg, R; Lee, J; Wolcott, A; Zhang, J; Terminello, L; van Buuren, T

    2009-10-27

    The exciton binding energy (EBE) in CdSe quantum dots (QDs) has been determined using x-ray spectroscopy. Using x-ray absorption and photoemission spectroscopy, the conduction band (CB) and valence band (VB) edge shifts as a function of particle size have been determined and combined to obtain the true band gap of the QDs (i.e. without and exciton). These values can be compared to the excitonic gap obtained using optical spectroscopy to determine the EBE. The experimental EBE results are compared with theoretical calculations on the EBE and show excellent agreement.

  12. Trace metals in gills of fish from the Arabian Gulf

    SciTech Connect

    Al-Yakoob, S.; Bahloul, M. ); Bou-Olayan, A.H.

    1994-11-01

    Complexation of metals by coordinate linkages with appropriate organic molecules in biological tissues is an important process involved in metal accumulation by aquatic organisms. Fish respiratory systems differ from all other systems because damage to gills has immediate impacts on the rest of the fish's body. Veer et al. observed significant correlation between gill-metal concentration and whole-body weight. More nickel is accumulated in gill tissue of the catfish (Clarias batrachus) than in the liver or intestine. More cadmium is accumulated in gill tissue of the fish Heteropneustes fossilis (Bloch) and Channa punctatus (Bloch) than in the liver or kidney. When exposed to lethal and sublethal concentrations of copper, gills of the freshwater fish Labeo rohita (Hamilton) showed the highest degree of copper accumulation. Petroleum and petrochemical industry wastes contribute significantly to metal enrichment of the Arabian Gulf marine environment. Because accumulation of metal ions is significant in gills, levels of Cd, Cr, Cu, Ni and Pb were investigated in gills of fish from potentially impacted areas along the western side of the Arabian Gulf after the 1991 oil-spill. 15 refs., 3 figs., 1 tab.

  13. Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations

    PubMed Central

    Bolon, Daniel; Clapham, Paul R.

    2016-01-01

    The conformation of HIV-1 envelope (Env) glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs) for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD) was investigated using a novel saturation mutagenesis approach. Single mutations identified, resulted in distinct trimer conformations affecting CD4bs exposure, the glycan shield and the TAD across diverse HIV-1 clades. Importantly, mutations that improve access to the CD4bs without exposing the immunodominant V3 loop were identified. The different trimer conformations identified will affect the specificity and breadth of nabs elicited in vivo and are important to consider in design of Env immunogens for vaccines. PMID:27820858

  14. CD14 mediates binding of high doses of LPS but is dispensable for TNF-α production.

    PubMed

    Borzęcka, Kinga; Płóciennikowska, Agnieszka; Björkelund, Hanna; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2013-01-01

    Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-α release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-α production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-α was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF α production induced by high concentrations of sLPS and rLPS can be limited.

  15. Modes of metal toxicity and impaired branchial ionoregulation in rainbow trout exposed to mixtures of Pb and Cd in soft water.

    PubMed

    Birceanu, Oana; Chowdhury, M Jasim; Gillis, Patricia L; McGeer, James C; Wood, Chris M; Wilkie, Michael P

    2008-09-29

    Models such as the Biotic Ligand Model (BLM) predict how natural organic matter (NOM) and competing ions (e.g., Ca(2+), H(+) and Na(+)) affect metal bioavailability and toxicity in aquatic organisms. However, such models focus upon individual metals, not metal mixtures. This study determined whether Pb and Cd interact at the gill of rainbow trout (Oncorhynchus mykiss) when trout were exposed to environmentally relevant concentrations of these metals (Cd<100 nmol L(-1); Pb<500 nmol L(-1)) in soft (<100 micromol Ca(2+)L(-1)), moderately acidic (pH 6.0) water. The 96-h LC50 for Pb was 482 nmol L(-1), indicating that Pb was one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, 6.7 nmol L(-1). Surprisingly, fish acclimated to soft water had multiple populations of Pb-gill and Cd-gill binding sites. A low capacity, high affinity population of Pb-gill binding sites had a B(max) of 18.2 nmol g(-1) wet weight (ww) and apparent K(Pb-gill)=7.05, but a second low affinity population could not be saturated up to free Pb concentrations approaching 4000 nmol L(-1). Two populations of Cd-gill binding sites were characterized: a high affinity, low capacity population with an apparent K(Cd-gill)=7.33 and B(max)=1.73 nmol g(-1) ww, and a low affinity, high capacity population with an apparent K(Cd-gill)=5.86, and B(max)=13.7 nmol g(-1) ww. At low concentrations, Cd plus Pb accumulation was less than additive because Cd out-competed Pb for gill binding sites, which were likely apical Ca(2+)-channels. While disturbances to Ca(2+) influx were caused by Cd alone, Pb alone had no effect. However, Pb exacerbated Cd-induced disturbances to Ca(2+) influx demonstrating that, although Pb- plus Cd-gill binding was less than additive due to competition, the effects (ionic disturbances) were more than additive (synergistic). Pb was also likely binding to intracellular targets, such as branchial carbonic anhydrase

  16. The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure

    PubMed Central

    1992-01-01

    The first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, Lys46, and Arg59, on the C, C', C", and D strands, respectively. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gp120. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction. PMID:1500858

  17. Quantifying Pb and Cd complexation by alginates and the role of metal binding on macromolecular aggregation.

    PubMed

    Lamelas, Cristina; Avaltroni, Fabrice; Benedetti, Marc; Wilkinson, Kevin J; Slaveykova, Vera I

    2005-01-01

    The Pb and Cd binding capacity of alginates were quantified by the determination of their complex stability constants and the concentration of complexing sites using H+, Pb2+, or Cd2+ selective electrodes in both static and dynamic titrations. Centrifugation filter devices (30 kDa filter cutoff), followed by inductively coupled plasma mass spectrometry (ICP-MS) measurements of lead or cadmium in the filtrates, were used to validate the results. The influence of ionic strength, pH, and the metal-to-alginate ratio was determined for a wide range of metal concentrations. Because of their polyelectrolytic properties, alginates may adopt different conformations depending on the physicochemistry of the medium, including the presence of metals. Therefore, molecular diffusion coefficients of the alginate were determined by fluorescence correlation spectroscopy under the same conditions of pH, ionic strength, and metal-to-alginate ratios that were used for the metal binding studies. The complexation and conformational properties of the alginate were related within the framework of the nonideal competitive adsorption isotherm (NICA) combined with a Donnan approach to account for both intrinsic and electrostatic contributions.

  18. Endogenous PMN sialidase activity exposes activation epitope on CD11b/CD18 which enhances its binding interaction with ICAM-1.

    PubMed

    Feng, Chiguang; Zhang, Lei; Almulki, Lama; Faez, Sepideh; Whitford, Melissa; Hafezi-Moghadam, Ali; Cross, Alan S

    2011-08-01

    Diapedesis is a dynamic, highly regulated process by which leukocytes are recruited to inflammatory sites. We reported previously that removal of sialyl residues from PMNs enables these cells to become more adherent to EC monolayers and that sialidase activity within intracellular compartments of resting PMNs translocates to the plasma membrane following activation. We did not identify which surface adhesion molecules were targeted by endogenous sialidase. Upon activation, β2 integrin (CD11b/CD18) on the PMN surface undergoes conformational change, which allows it to bind more tightly to the ICAM-1 and ICAM-2 on the EC surface. Removal of sialyl residues from CD18 and CD11b, by exogenous neuraminidase or mobilization of PMN sialidase, unmasked activation epitopes, as detected by flow cytometry and enhanced binding to ICAM-1. One sialidase isoform, Neu1, colocalized with CD18 on confocal microscopy. Using an autoperfused microflow chamber, desialylation of immobilized ICAM-1 enhanced leukocyte arrest in vivo. Further, treatment with a sialidase inhibitor in vivo reversed endotoxin-induced binding of leukocytes to ICAM-1, thereby suggesting a role for leukocyte sialidase in the cellular arrest. These data suggest that PMN sialidase could be a physiologic source of the enzymatic activity that removes sialyl residues on β2 integrin and ICAM-1, resulting in their enhanced interaction. Thus, PMN sialidase may be an important regulator of the recruitment of these cells to inflamed sites.

  19. Lineage-specific differences between human and simian immunodeficiency virus regulation of gp120 trimer association and CD4 binding.

    PubMed

    Finzi, Andrés; Pacheco, Beatriz; Xiang, Shi-Hua; Pancera, Marie; Herschhorn, Alon; Wang, Liping; Zeng, Xing; Desormeaux, Anik; Kwong, Peter D; Sodroski, Joseph

    2012-09-01

    Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.

  20. Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

    PubMed

    Kneidl, Jessica; Mysore, Vijayashree; Geraci, Jennifer; Tuchscherr, Lorena; Löffler, Bettina; Holzinger, Dirk; Roth, Johannes; Barczyk-Kahlert, Katarzyna

    2014-03-01

    Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

  1. Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis.

    PubMed

    Della Torre, Camilla; Balbi, Teresa; Grassi, Giacomo; Frenzilli, Giada; Bernardeschi, Margherita; Smerilli, Arianna; Guidi, Patrizia; Canesi, Laura; Nigro, Marco; Monaci, Fabrizio; Scarcelli, Vittoria; Rocco, Lucia; Focardi, Silvano; Monopoli, Marco; Corsi, Ilaria

    2015-10-30

    We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media.

  2. The crystal structure of human CD21: Implications for Epstein-Barr virus and C3d binding.

    PubMed

    Prota, Andrea E; Sage, David R; Stehle, Thilo; Fingeroth, Joyce D

    2002-08-06

    Human complement receptor type 2 (CD21) is the cellular receptor for Epstein-Barr virus (EBV), a human tumor virus. The N-terminal two short consensus repeats (SCR1-SCR2) of the receptor interact with the EBV glycoprotein gp350/220 and also with the natural CD21 ligand C3d. Here we present the crystal structure of the CD21 SCR1-SCR2 fragment in the absence of ligand and demonstrate that it is able to bind EBV. Based on a functional analysis of wild-type and mutant CD21 and molecular modeling, we identify a likely region for EBV attachment and demonstrate that this region is not involved in the interaction with C3d. A comparison with the previously determined structure of CD21 SCR1-SCR2 in complex with C3d shows that, in both cases, CD21 assumes compact V-shaped conformations. However, our analysis reveals a surprising degree of flexibility at the SCR1-SCR2 interface, suggesting interactions between the two domains are not specific. We present evidence that the V-shaped conformation is induced by deglycosylation of the protein, and that physiologic glycosylation of CD21 would result in a more extended conformation, perhaps with additional epitopes for C3d binding.

  3. Differential regulation of monocyte cytokine release by αV and β(2) integrins that bind CD23.

    PubMed

    Edkins, Adrienne L; Borland, Gillian; Acharya, Mridu; Cogdell, Richard J; Ozanne, Bradford W; Cushley, William

    2012-06-01

    The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVβ(3), αVβ(5), αMβ(2) and αXβ(2), but it is unclear which of these four integrins most acutely regulates sCD23-driven cytokine release. The hypothesis that ligation of different sCD23-binding integrins promoted release of distinct subsets of cytokines was tested. Lipopolysaccharide (LPS) and sCD23 promoted release of distinct groups of cytokines from the THP-1 model cell line. The sCD23-driven cytokine release signature was characterized by elevated amounts of RANTES (CCL5) and a striking increase in interleukin-8 (IL-8; CXCL8) secretion, but little release of macrophage inflammatory protein 1β (MIP-1β; CCL4). Antibodies to αVβ(3) or αXβ(2) both promoted IL-8 release, consistent with the sCD23-driven pattern, but both also evoked strong MIP-1β secretion; simultaneous ligation of these two integrins further increased cytokine secretion but did not alter the pattern of cytokine output. In both model cell lines and primary tissue, integrin-mediated cytokine release was more pronounced in immature monocyte cells than in mature cells. The capacity of anti-integrin monoclonal antibodies to elicit a cytokine release response is epitope-dependent and also reflects the differentiation state of the cell. Although a pattern of cytokine release identical to that provoked by sCD23 could not be elicited with any individual anti-integrin monoclonal antibody, αXβ(2) and αVβ(3) appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMβ(2) or αVβ(5).

  4. Envelope residue 375 substitutions in simian-human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques.

    PubMed

    Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D; Farzan, Michael; Chertova, Elena; Keele, Brandon F; Estes, Jacob D; Lifson, Jeffrey D; Doms, Robert W; Montefiori, David C; Haynes, Barton F; Sodroski, Joseph G; Kwong, Peter D; Hahn, Beatrice H; Shaw, George M

    2016-06-14

    Most simian-human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants-S, M, Y, H, W, or F-that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env-rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors.

  5. Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

    PubMed Central

    Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H.; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G.; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S. Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D.; Farzan, Michael; Chertova, Elena; Keele, Brandon F.; Estes, Jacob D.; Lifson, Jeffrey D.; Doms, Robert W.; Montefiori, David C.; Haynes, Barton F.; Sodroski, Joseph G.; Kwong, Peter D.; Hahn, Beatrice H.; Shaw, George M.

    2016-01-01

    Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors. PMID:27247400

  6. γ-Tilmanocept, a New Radiopharmaceutical Tracer for Cancer Sentinel Lymph Nodes, Binds to the Mannose Receptor (CD206)

    PubMed Central

    Azad, Abul K.; Rajaram, Murugesan V. S.; Metz, Wendy L.; Cope, Frederick O.; Blue, Michael S.; Vera, David R.

    2015-01-01

    γ-Tilmanocept (99mTc-labeled-tilmanocept or [99mTc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR+ cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept–binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases. PMID:26202986

  7. Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4†

    PubMed Central

    Yang, Quan-en; Stephen, Andrew G.; Adelsberger, Joseph W.; Roberts, Paula E.; Zhu, Weimin; Currens, Michael J.; Feng, Yaxiong; Crise, Bruce J.; Gorelick, Robert J.; Rein, Alan R.; Fisher, Robert J.; Shoemaker, Robert H.; Sei, Shizuko

    2005-01-01

    The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4+ cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions. PMID:15857997

  8. Insight into the modified Ibalizumab-human CD4 receptor interactions: using a computational binding free energy approach.

    PubMed

    Wang, Yeng-Tseng; Chuang, Lea-Yea

    2015-01-01

    Antibody drugs are very useful tools for the treatment of many chronic diseases. Recently, however, patients and doctors have encountered the problem of drug resistance. How to improve the affinity of antibody drugs has therefore become a pressing issue. Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1. This study investigates the mutation residues of the complementarity determining regions of Ibalizumab. We propose using the wild and mutations of Ibalizumab-human CD4 receptor complex structures, molecular dynamics techniques, alanine-scanning mutagenesis calculations and solvated interaction energies methods to predict the binding free energy of the Ibalizumab-human CD4 receptor complex structures. This work found that revealed three key positions (31th, 32th and 33th in HCDR-1) of the residues may play an important role in Ibalizumab-human CD4 receptor complex interactions. Therefore, bioengineering substitutions of the three key positions and increasing number of intermolecular interactions (HCDR-1 of Ibalizumab/human CD4 receptor) might improve the binding affinities of this complex structure.

  9. Insight into the modified Ibalizumab-human CD4 receptor interactions: using a computational binding free energy approach

    NASA Astrophysics Data System (ADS)

    Wang, Yeng-Tseng; Chuang, Lea-Yea

    2015-01-01

    Antibody drugs are very useful tools for the treatment of many chronic diseases. Recently, however, patients and doctors have encountered the problem of drug resistance. How to improve the affinity of antibody drugs has therefore become a pressing issue. Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1. This study investigates the mutation residues of the complementarity determining regions of Ibalizumab. We propose using the wild and mutations of Ibalizumab-human CD4 receptor complex structures, molecular dynamics techniques, alanine-scanning mutagenesis calculations and solvated interaction energies methods to predict the binding free energy of the Ibalizumab-human CD4 receptor complex structures. This work found that revealed three key positions (31th, 32th and 33th in HCDR-1) of the residues may play an important role in Ibalizumab-human CD4 receptor complex interactions. Therefore, bioengineering substitutions of the three key positions and increasing number of intermolecular interactions (HCDR-1 of Ibalizumab/human CD4 receptor) might improve the binding affinities of this complex structure.

  10. Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

    PubMed Central

    Gil-Moreno, Selene; Jiménez-Martí, Elena; Palacios, Òscar; Zerbe, Oliver; Dallinger, Reinhard; Capdevila, Mercè; Atrian, Sílvia

    2015-01-01

    Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. PMID:26703589

  11. Biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of chondrocyte adhesion to scaffolds.

    PubMed

    Lin, Hong; Zhou, Jian; Shen, Longxiang; Ruan, Yuhui; Dong, Jian; Guo, Changan; Chen, Zhengrong

    2014-04-01

    The clinical need for improved treatment options for patients with cartilage injuries has motivated tissue-engineering studies aimed at the in vitro generation of cell-based implants with functional properties. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to the scaffold. In the present study, chondrocyte-scaffold constructs were engineered by planting porcine chondrocytes into nonporous chitosan membranes and 3D porous chitosan scaffolds that were treated with or without biotin-conjugated anti-CD44 antibody-avidin binding system and avidin-biotin binding system. The spreading area, cell exfoliation rates, cell proliferation rates, histological analysis, DNA and glycosaminoglycan (GAG) content, and mRNA expression were investigated to evaluate the efficiency of biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of cell adhesion to scaffolds in the cartilage tissue. The results showed that the biotin-conjugated anti-CD44 antibody-avidin binding system improved cell adhesion to scaffolds effectively. These studies suggest that this binding system has the potential to provide improved tissue-engineered cartilage for clinical applications.

  12. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    SciTech Connect

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  13. The Cd(II)-binding abilities of recombinant Quercus suber metallothionein: bridging the gap between phytochelatins and metallothioneins.

    PubMed

    Domènech, Jordi; Orihuela, Rubén; Mir, Gisela; Molinas, Marisa; Atrian, Sílvia; Capdevila, Mercè

    2007-08-01

    In this work, we have analyzed both at stoichiometric and at conformational level the Cd(II)-binding features of a type 2 plant metallothionein (MT) (the cork oak, Quercus suber, QsMT). To this end four peptides, the wild-type QsMT and three constructs previously engineered to characterize its Zn(II)- and Cu(I)-binding behaviour, were heterologously produced in Escherichia coli cultures supplemented with Cd(II), and the corresponding complexes were purified up to homogeneity. The Cd(II)-binding ability of these recombinant peptides was determined through the chemical, spectroscopic and spectrometric characterization of the recovered clusters. Recombinant synthesis of the four QsMT peptides in cadmium-rich media rendered complexes with a higher metal content than those obtained from zinc-supplemented cultures and, consequently, the recovered Cd(II) species are nonisostructural to those of Zn(II). Also of interest is the fact that three out of the four peptides yielded recombinant preparations that included S(2-)-containing Cd(II) complexes as major species. Subsequently, the in vitro Zn(II)/Cd(II) replacement reactions were studied, as well as the in vitro acid denaturation and S(2-) renaturation reactions. Finally, the capacity of the four peptides for preventing cadmium deleterious effects in yeast cells was tested through complementation assays. Consideration of all the results enables us to suggest a hairpin folding model for this typical type 2 plant Cd(II)-MT complex, as well as a nonnegligible role of the spacer in the detoxification function of QsMT towards cadmium.

  14. Molecular docking guided structure based design of symmetrical N,N'-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors.

    PubMed

    Sivan, Sree Kanth; Vangala, Radhika; Manga, Vijjulatha

    2013-08-01

    Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N'-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations.

  15. Modulation of NMDA channel gating by Ca2+ and Cd2+ binding to the external pore mouth

    PubMed Central

    Tu, Ya-Chi; Yang, Ya-Chin; Kuo, Chung-Chin

    2016-01-01

    NMDA receptor channels are characterized by high Ca2+ permeability. It remains unclear whether extracellular Ca2+ could directly modulate channel gating and control Ca2+ influxes. We demonstrate a pore-blocking site external to the activation gate for extracellular Ca2+ and Cd2+, which has the same charge and radius as Ca2+ but is impermeable to the channel. The apparent affinity of Cd2+ or Ca2+ is higher toward the activated (a steady-state mixture of the open and desensitized, probably chiefly the latter) than the closed states. The blocking effect of Cd2+ is well correlated with the number of charges in the DRPEER motif at the external pore mouth, with coupling coefficients close to 1 in double mutant cycle analyses. The effect of Ca2+ and especially Cd2+ could be allosterically affected by T647A mutation located just inside the activation gate. A prominent “hook” also develops after wash-off of Cd2+ or Ca2+, suggesting faster unbinding rates of Cd2+ and Ca2+ with the mutation. We conclude that extracellular Ca2+ or Cd2+ directly binds to the DRPEER motif to modify NMDA channel activation (opening as well as desensitization), which seems to involve essential regional conformational changes centered at the bundle crossing point A652 (GluN1)/A651(GluN2). PMID:27848984

  16. CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

    PubMed Central

    Martínez-Muñoz, Laura; Barroso, Rubén; Dyrhaug, Sunniva Y.; Navarro, Gemma; Lucas, Pilar; Soriano, Silvia F.; Vega, Beatriz; Costas, Coloma; Muñoz-Fernández, M. Ángeles; Santiago, César; Frade, José Miguel Rodríguez; Franco, Rafael; Mellado, Mario

    2014-01-01

    CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4+ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention. PMID:24778234

  17. Cystein 402 of HIV gp 120 is essential for CD4-binding and resistance of gp 120 to intracellular degradation.

    PubMed

    Hemming, A; Bolmstedt, A; Flodby, P; Lundberg, L; Gidlund, M; Wigzell, H; Olofsson, S

    1989-01-01

    A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp 120 was excised from an SV40-based expression vector containing gp 160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys402 was substituted for a serine, and metabolically labelled with [3H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp 120/gp 160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp 160, whereas gp 120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80-105k, suggesting increased sensitivity of mutant env gene products to proteolysis after cleavage to gp 120. Wild type gp 120 and gp 160 bound to CD4, whereas neither gp 160 nor gp 120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys402, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability of env gene products and for (ii) the physical stability of gp 120.

  18. Link protein hyaluronan-binding motif abrogates CD44-hyaluronan-mediated leukemia-liver cell adhesion.

    PubMed

    Chen, Jing; Li, Na; Li, Gongchu

    2013-05-01

    The liver is a frequent site for the metastasis of cancer cells originating from other sites. Leukemic liver metastasis is associated with poor prognosis. The ligation of CD44 with hyaluronan (HA) has been shown to contribute to the drug resistance of leukemic cells. In this study, a link protein HA-binding motif was genetically fused with enhanced green fluorescence protein (EGFP) to generate an EGFP-L fusion protein. Furthermore, a coculture system was established to investigate the interaction of leukemic cells with liver cells. CD44-positive Kasumi-1, but not CD44-negative HL-60 cells, were observed to adhere to the liver cell line L02. This cell-cell adhesion was significantly blocked by HA, indicating that Kasumi-L02 cell adhesion was mediated by the CD44-HA interaction. Compared to EGFP, EGFP-L fusion protein bound to L02 and BEL7404 liver cells. EGFP-L partially abrogated the Kasumi-L02 adhesion, suggesting that the link protein-binding motif is able to inhibit CD44-HA-mediated leukemia-liver adhesion. These results may help provide insight into novel therapeutic methods for leukemic patients diagnosed with liver metastasis.

  19. Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

    PubMed

    Stax, Martijn J; Mouser, Emily E I M; van Montfort, Thijs; Sanders, Rogier W; de Vries, Henry J C; Dekker, Henk L; Herrera, Carolina; Speijer, Dave; Pollakis, Georgios; Paxton, William A

    2015-01-01

    Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

  20. Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.

    PubMed

    Hughes, Timothy R; Ross, Kirsty S; Cowan, Graeme J M; Sivasankar, Baalasubramanian; Harris, Claire L; Mitchell, Timothy J; Morgan, B Paul

    2009-04-01

    The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

  1. Soluble CD14 subtype presepsin (sCD14-ST) and lipopolysaccharide binding protein (LBP) in neonatal sepsis: new clinical and analytical perspectives for two old biomarkers.

    PubMed

    Mussap, Michele; Noto, Antonio; Fravega, Marco; Fanos, Vassilios

    2011-10-01

    Several biochemical markers have been proposed over the past years to manage critically ill newborns with acute inflammation and sepsis. The state of the art in diagnosing and monitoring neonatal sepsis, severe sepsis and septic shock consists of the measurement of plasma C-reactive protein (CRP) and procalcitonin (PCT) at the onset and in the course of the disease. CRP and PCT in combination are clinically significant in diagnosing and monitoring septic newborns; however, CRP and PCT have a very limited value for risk stratification and in predicting outcome. The availability of commercial methods for the automated measurement of the soluble CD14 subtype presepsin (sCD14-ST) and lipopolysaccharide binding protein (LBP) represent a challenge for the evaluation in clinical practice of reliable markers of neonatal sepsis, specifically for the very early diagnosis, the classification into class of severity, and the prediction of complications and death.

  2. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth

    SciTech Connect

    Huang, Jinghe; Kang, Byong H.; Ishida, Elise; Zhou, Tongqing; Griesman, Trevor; Sheng, Zizhang; Wu, Fan; Doria-Rose, Nicole A.; Zhang, Baoshan; McKee, Krisha; O’Dell, Sijy; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Schramm, Chaim A.; Zheng, Anqi; Joyce, M.  Gordon; Asokan, Mangaiarkarasi; Ransier, Amy; Darko, Sam; Migueles, Stephen A.; Bailer, Robert T.; Louder, Mark K.; Alam, S.  Munir; Parks, Robert; Kelsoe, Garnett; Von Holle, Tarra; Haynes, Barton F.; Douek, Daniel C.; Hirsch, Vanessa; Seaman, Michael S.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2016-11-01

    Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.

  3. Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

    PubMed

    Wah, D A; Romero, A; Gallego del Sol, F; Cavada, B S; Ramos, M V; Grangeiro, T B; Sampaio, A H; Calvete, J J

    2001-07-20

    Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure

  4. MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma.

    PubMed

    Gebhard, Anthony W; Jain, Priyesh; Nair, Rajesh R; Emmons, Michael F; Argilagos, Raul F; Koomen, John M; McLaughlin, Mark L; Hazlehurst, Lori A

    2013-11-01

    Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101-induced cell death. Mechanistically, we show that MTI-101-induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma.

  5. MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma

    PubMed Central

    Gebhard, Anthony W.; Jain, Priyesh; Nair, Rajesh R.; Emmons, Michael F.; Argilagos, Raul F.; Koomen, John M.; McLaughlin, Mark L.; Hazlehurst, Lori A.

    2013-01-01

    Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma (MM) cell lines. Due to the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels and failed to activate caspase 3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and LC-MS/MS analysis to identify binding partners of MTI-101. Using this approach CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in MM cell lines, indicating that MM cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101 induced cell death. Mechanistically, we show that MTI-101 induced cell death occurs via a Rip1, Rip3 or Drp1 dependent and independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. PMID:24048737

  6. Human leucocyte antigen class I‐redirected anti‐tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells

    PubMed Central

    Tan, M. P.; Dolton, G. M.; Gerry, A. B.; Brewer, J. E.; Bennett, A. D.; Pumphrey, N. J.; Jakobsen, B. K.

    2016-01-01

    Summary CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour‐specific CD4+ T cells occur in low frequency, express relatively low‐affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour‐specific HLA class I‐restricted TCRs prior to adoptive transfer. The lack of help from the co‐receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild‐type and a range of affinity‐enhanced TCRs specific for the HLA A*0201‐restricted NY‐ESO‐1‐ and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement. PMID:27324616

  7. The role of higher-order protein structure in supporting binding by heteroclitic monoclonal antibodies: the monoclonal antibody KIM185 to CD18 also binds C4-binding protein.

    PubMed

    Gjelstrup, Louise Carstensen; Andersen, Stig Henrik; Petersen, Steen Vang; Enghild, Jan J; Blom, Anna M; Vorup-Jensen, Thomas; Thiel, Steffen

    2011-10-01

    Heteroclitic monoclonal antibodies are characterized by the ability to bind multiple epitopes with little or no similarity. Such antibodies have been reported earlier, but insight into to the molecular basis of this propensity is limited. Here we report that the KIM185 antibody to human CD18 reacts with the plasma protein C4b-binding protein (C4BP). This was revealed during affinity purification procedures where human serum was incubated with surfaces coated with monoclonal antibodies to CD18. Other monoclonal antibodies to CD18 (KIM127 and TS1/18) showed no such interaction with C4BP. We constructed a sandwich-type time-resolved immunofluorometric assay using KIM185 both as capture and developing antibody. By use of proteolytic fragments of KIM185 and recombinant deletion mutants of C4BP the interaction sites were mapped to the variable region of KIM185 and the oligomerization domain of C4BP, respectively. C4BP is a large oligomeric plasma protein that binds activated complement factor C4b and other endogenous ligands as well as microorganisms. By use of the recent crystallographic data on the structure of CD11c/CD18 and prediction of the secondary structure of the C4BP oligomerization domain, we show that epitopes bound by KIM185 in these proteins are unlikely to share any major structural similarity. However, both antigens may form oligomers that would enable avid binding by the antibody. Our report points to the astonishing ability of heteroclitic antibodies to accommodate the binding of multiple proteins with no or little structural similarity within the confined space of the variable regions.

  8. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    PubMed

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  9. The Effects of Cadmium Exposure on the Oxidative State and Cell Death in the Gill of Freshwater Crab Sinopotamon henanense

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Shen, Qingqing; Wang, Qian; Liu, Dongmei; Li, Jing; Wang, Lan

    2013-01-01

    We studied here the short-term toxicity effects of Cd on the oxidative state and cell death in the gill of freshwater crab Sinopotamon henanense. Crabs were exposed to Cd that resulted in Cd accumulation and a significant increase in the metallothionein (MT) level in the gill, but MT level increased disproportionally compared to the Cd accumulation with an extension of exposure time. Significant changes in the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were observed. An increase in the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) was detected that will cause oxidative stress. Histological abnormalities of the gills were discovered, including the expansion of gill cavity, a decrease in the numbers of connection of the upper and the lower of the gill lamellae and epithelial cells, and an increase in the number of hemocytes. The results of a TUNEL test and transmission electron microscope (TEM) showed that more gill cells had apoptotic characteristics after 48 h of Cd treatment compared to the control, but epithelial cell necrosis and inflammatory response appeared only after 72 h. It was concluded that (1) Cd induced the ROS production and accumulation through inhibiting antioxidant enzyme activities and exceeding the saturation values of MT binging; (2) Cd led to lipid peroxidation and histopathological alternations; and (3) Cd induced apoptotic response at short time exposure, followed by necrotic features and inflammatory reaction after longer time exposure. PMID:23737962

  10. Crystal structures of bovine CD1d reveal altered αGalCer presentation and a restricted A' pocket unable to bind long-chain glycolipids.

    PubMed

    Wang, Jing; Guillaume, Joren; Pauwels, Nora; Van Calenbergh, Serge; Van Rhijn, Ildiko; Zajonc, Dirk M

    2012-01-01

    NKT cells play important roles in immune surveillance. They rapidly respond to pathogens by detecting microbial glycolipids when presented by the non-classical MHC I homolog CD1d. Previously, ruminants were considered to lack NKT cells due to the lack of a functional CD1D gene. However, recent data suggest that cattle express CD1d with unknown function. In an attempt to characterize the function of bovine CD1d, we assessed the lipid binding properties of recombinant Bos taurus CD1d (boCD1d) in vitro. BoCD1d is able to bind glycosphingolipids (GSLs) with fatty acid chain lengths of C₁₈, while GSLs with fatty acids of C₂₄ do not bind. Crystal structures of boCD1d bound to a short-chain C₁₂-di-sulfatide antigen, as well as short-chain C₁₆-αGalCer revealed that the Á pocket of boCD1d is restricted in size compared to that of both mouse and human CD1d, explaining the inability of long chain GSL's to bind to boCD1d. Moreover, while di-sulfatide is presented similarly compared to the presentation of sulfatide by mouse CD1d, αGalCer is presented differently at the cell surface, due to an amino acid Asp151Asn substitution that results in loss of intimate contacts between the αGalCer headgroup and CD1d. The altered αGalCer presentation by boCD1d also explains its lack of cross-activation of mouse iNKT cells and raises the interesting question of the nature and function of bovine lipid-reactive T cells.

  11. Crystal Structures of Bovine CD1d Reveal Altered αGalCer Presentation and a Restricted A’ Pocket Unable to Bind Long-Chain Glycolipids

    PubMed Central

    Wang, Jing; Guillaume, Joren; Pauwels, Nora; Van Calenbergh, Serge; Van Rhijn, Ildiko; Zajonc, Dirk M.

    2012-01-01

    NKT cells play important roles in immune surveillance. They rapidly respond to pathogens by detecting microbial glycolipids when presented by the non-classical MHC I homolog CD1d. Previously, ruminants were considered to lack NKT cells due to the lack of a functional CD1D gene. However, recent data suggest that cattle express CD1d with unknown function. In an attempt to characterize the function of bovine CD1d, we assessed the lipid binding properties of recombinant Bos taurus CD1d (boCD1d) in vitro. BoCD1d is able to bind glycosphingolipids (GSLs) with fatty acid chain lengths of C18, while GSLs with fatty acids of C24 do not bind. Crystal structures of boCD1d bound to a short-chain C12-di-sulfatide antigen, as well as short-chain C16-αGalCer revealed that the Á pocket of boCD1d is restricted in size compared to that of both mouse and human CD1d, explaining the inability of long chain GSL’s to bind to boCD1d. Moreover, while di-sulfatide is presented similarly compared to the presentation of sulfatide by mouse CD1d, αGalCer is presented differently at the cell surface, due to an amino acid Asp151Asn substitution that results in loss of intimate contacts between the αGalCer headgroup and CD1d. The altered αGalCer presentation by boCD1d also explains its lack of cross-activation of mouse iNKT cells and raises the interesting question of the nature and function of bovine lipid-reactive T cells. PMID:23110152

  12. Simultaneous determination of Ca, Cu, Ni, Zn and Cd binding strengths with fulvic acid fractions by Schubert's method

    USGS Publications Warehouse

    Brown, G.K.; MacCarthy, P.; Leenheer, J.A.

    1999-01-01

    The equilibrium binding of Ca2+, Ni2+, Cd2+, Cu2+ and Zn2+ with unfractionated Suwannee river fulvic acid (SRFA) and an enhanced metal binding subfraction of SRFA was measured using Schubert's ion-exchange method at pH 6.0 and at an ionic strength (??) of 0.1 (NaNO3). The fractionation and subfractionation were directed towards obtaining an isolate with an elevated metal binding capacity or binding strength as estimated by Cu2+ potentiometry (ISE). Fractions were obtained by stepwise eluting an XAD-8 column loaded with SRFA with water eluents of pH 1.0 to pH 12.0. Subfractions were obtained by loading the fraction eluted from XAD-8 at pH 5.0 onto a silica gel column and eluting with solvents of increasing polarity. Schuberts ion exchange method was rigorously tested by measuring simultaneously the conditional stability constants (K) of citric acid complexed with the five metals at pH 3.5 and 6.0. The logK of SRFA with Ca2+, Ni2+, Cd2+, Cu2+ and Zn2+ determined simultaneously at pH 6.0 follow the sequence of Cu2+>Cd2+>Ni2+>Zn2+>Ca2+ while all logK values increased for the enhanced metal binding subfraction and followed a different sequence of Cu2+>Cd2+>Ca2+>Ni2+>Zn2+. Both fulvic acid samples and citric acid exhibited a 1:1 metal to ligand stochiometry under the relatively low metal loading conditions used here. Quantitative 13C nuclear magnetic resonance spectroscopy showed increases in aromaticity and ketone content and decreases in aliphatic carbon for the elevated metal binding fraction while the carboxyl carbon, and elemental nitrogen, phosphorus, and sulfur content did not change. The more polar, elevated metal binding fraction did show a significant increase in molecular weight over the unfractionated SRFA. Copyright (C) 1999 Elsevier Science B.V.

  13. Combined application of a laser ablation-ICP-MS assay for screening and ESI-FTICR-MS for identification of a Cd-binding protein in Spinacia oleracea L. after exposure to Cd.

    PubMed

    Polatajko, Aleksandra; Feldmann, Ingo; Hayen, Heiko; Jakubowski, Norbert

    2011-10-01

    We have studied the binding of the toxic element Cd to plant proteins and have used for this purpose spinach (Spinacia oleracea L.) plants treated with 50 μM Cd(II) as a model system. Laser ablation ICP-MS has been applied for the screening of Cd-binding proteins after separation by native anodal polyacrylamide gel electrophoresis (AN-PAGE) and electroblotting onto membranes. The main Cd-carrying protein band was isolated and investigated by nano-electrospray ionization-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry after tryptic digestion. By this procedure, the main Cd-binding protein was identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The latter enzyme has been discussed in the literature to be affected in its activity by oxidative stress induced by Cd. However, in this paper it is demonstrated for the first time that RuBisCO directly binds Cd and thus may be directly altered by this toxic element. A commercially available protein standard was used to verify direct binding of Cd(II) to the protein, even without metabolisation. The resulting metal-protein complex was shown to be stable enough to survive AN-PAGE separation and electroblotting. By the use of size exclusion chromatography coupled with ICP-MS it was demonstrated that the RuBisCO protein standard shows similar metal binding properties to Cd. Furthermore, essential elements such as Mn(II), Fe(II) and Cu(II), which are known to possibly replace the RuBisCO activator Mg(II), were investigated in addition to Zn(II). Again, similar binding properties in comparison to the plant protein were observed.

  14. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    SciTech Connect

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey  E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan  A.; Alam, S.  Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia  D.; Kamanga, Gift; Cohen, Myron  S.; Sam, Noel  E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy  L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw  K.; Mascola, John  R.; Hahn, Beatrice H.; Shaw, George  M.; Sodroski, Joseph  G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  15. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    DOE PAGES

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; ...

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

  16. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

    PubMed

    Moody, M Anthony; Gao, Feng; Gurley, Thaddeus C; Amos, Joshua D; Kumar, Amit; Hora, Bhavna; Marshall, Dawn J; Whitesides, John F; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan A; Alam, S Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia D; Kamanga, Gift; Cohen, Myron S; Sam, Noel E; Kapiga, Saidi; Gray, Elin S; Tumba, Nancy L; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw K; Mascola, John R; Hahn, Beatrice H; Shaw, George M; Sodroski, Joseph G; Liao, Hua-Xin; Montefiori, David C; Hraber, Peter T; Korber, Bette T; Haynes, Barton F

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  17. Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2.

    PubMed

    Manchester, M; Gairin, J E; Patterson, J B; Alvarez, J; Liszewski, M K; Eto, D S; Atkinson, J P; Oldstone, M B

    1997-06-23

    Measles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchester et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2303-2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37-56) and another within SCR2 (amino acids 85-104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2.

  18. Influence of Cd 2+, Hg 2+ and Pb 2+ on (+)-catechin binding to bovine serum albumin studied by fluorescence spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Peng, Mijun; Shi, Shuyun; Zhang, Yuping

    2012-01-01

    The effect of heavy metal ions, Cd 2+, Hg 2+ and Pb 2+ on (+)-catechin binding to bovine serum albumin (BSA) has been investigated by spectroscopic methods. The results indicated that the presence of heavy metal ions significantly affected the binding modes and binding affinities of (+)-catechin to BSA, and the effects depend on the types of heavy metal ion. One binding mode was found for (+)-catechin with and without Cd 2+, while two binding modes - a weaker one at low concentration and a stronger one at high concentration were found for (+)-catechin in the presence of Hg 2+ and Pb 2+. The presence of Cd 2+ decreased the binding affinities of (+)-catechin for BSA by 20.5%. The presence of Hg 2+ and Pb 2+ decreased the binding affinity of (+)-catechin for BSA by 8.9% and 26.7% in lower concentration, respectively, and increased the binding affinity of (+)-catechin for BSA by 5.2% and 9.2% in higher concentration, respectively. The changed binding affinity and binding distance of (+)-catechin for BSA in the presence of Cd 2+, Hg 2+ and Pb 2+ were mainly because of the conformational change of BSA induced by heavy metal ions. However, the quenching mechanism for (+)-catechin to BSA was based on static quenching combined with non-radiative energy transfer irrespective of the absence or presence of heavy metal ions.

  19. Mutations in CD2BP1 disrupt binding to PTP PEST and are responsible for PAPA syndrome, an autoinflammatory disorder.

    PubMed

    Wise, Carol A; Gillum, Joseph D; Seidman, Christine E; Lindor, Noralane M; Veile, Rose; Bashiardes, Stavros; Lovett, Michael

    2002-04-15

    PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne, OMIM #604416) and familial recurrent arthritis (FRA) are rare inherited disorders of early onset, primarily affecting skin and joint tissues. Recurring inflammatory episodes lead to accumulation of sterile, pyogenic, neutrophil-rich material within the affected joints, ultimately resulting in significant destruction. We recently localized the genes for PAPA syndrome and FRA to chromosome 15q and suggested that they are the same disorder. We have now established this by the identification of co-segregating disease-causing mutations in the CD2-binding protein 1 (CD2BP1; GenBank accession no XM 044569) gene in the two reported families with this disorder. E250Q or A230T amino acid substitutions occur within a domain highly homologous to yeast cleavage furrow-associated protein CDC15. CD2BP1 and its murine ortholog, proline-serine-threonine phosphatase interacting protein (PSTPIP1), are adaptor proteins known to interact with PEST-type protein tyrosine phosphatases (PTP). Yeast two-hybrid assays demonstrate severely reduced binding between PTP PEST and both the E250Q and A230T mutant proteins. Previous evidence supports the integral role of CD2BP1 and its interacting proteins in actin reorganization during cytoskeletal-mediated events. We hypothesize that the disease-causing mutations that we have identified compromise physiologic signaling necessary for the maintenance of proper inflammatory response. Accordingly we suggest classification of PAPA syndrome as an autoinflammatory disease. This CD2BP1-mediated biochemical pathway(s) may function in common inflammatory disorders with apparent etiological overlap, such as rheumatoid arthritis and inflammatory bowel disease.

  20. Comparative experiments of graphene covalently and physically binding CdSe quantum dots to enhance the electron transport in flexible photovoltaic devices.

    PubMed

    Jung, Mi-Hee; Chu, Moo-Jung

    2014-08-07

    In this research, we prepared composite films via covalent coupling of CdSe quantum dots (QDs) to graphene through the direct binding of aryl radicals to the graphene surface. To compare the carrier transport with the CdSe aryl binding graphene film, we prepared CdSe pyridine capping graphene films through the pi-pi interactions of noncovalent bonds between the graphene and pyridine molecules. The photovoltaic devices were fabricated from the two hybrid films using the electrophoretic deposition method on flexible substrates. Even though the two hybrid films have the same amount of QDs and graphene, time-resolved fluorescence emission decay results show that the emission lifetime of the CdSe aryl group binding graphene film is significantly shorter than that of the pyridine capping CdSe-graphene. The quantum efficiency and photocurrent density of the device fabricated from CdSe aryl binding graphene were also higher than those of the device fabricated from pyridine capping CdSe-graphene. These results indicated that the carrier transport of the QD-graphene system is not related to the additive effect from the CdSe and graphene components but rather is a result of the unique interactions between the graphene and QDs. We could expect that these results can be useful in designing QD-graphene composite materials, which are applied in photovoltaic devices.

  1. An x-ray absorption spectroscopy study of Cd binding onto a halophilic archaeon

    NASA Astrophysics Data System (ADS)

    Showalter, Allison R.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.; Bunker, Bruce A.

    2016-05-01

    X-ray absorption spectroscopy (XAS) and cadmium (Cd) isotherm experiments determine how Cd adsorbs to the surface of halophilic archaeon Halobacterium noricense. This archaeon, isolated from the Waste Isolation Pilot Plant (WIPP) near Carlsbad, New Mexico could be involved with the transport of toxic metals stored in the transuranic waste in the salt mine. The isotherm experiments show that adsorption is relatively constant across the tolerable pH range for H. noricense. The XAS results indicate that Cd adsorption occurs predominately via a sulfur site, most likely sulfhydryl, with the same site dominating all measured pH values.

  2. Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4(+) T Cells.

    PubMed

    St Gelais, Corine; Roger, Jonathan; Wu, Li

    2015-08-01

    HIV-1 interacts with numerous cellular proteins during viral replication. Identifying such host proteins and characterizing their roles in HIV-1 infection can deepen our understanding of the dynamic interplay between host and pathogen. We previously identified non-POU domain-containing octamer-binding protein (NonO or p54nrb) as one of host factors associated with catalytically active preintegration complexes (PIC) of HIV-1 in infected CD4(+) T cells. NonO is involved in nuclear processes including transcriptional regulation and RNA splicing. Although NonO has been identified as an HIV-1 interactant in several recent studies, its role in HIV-1 replication has not been characterized. We investigated the effect of NonO on the HIV-1 life cycle in CD4(+) T cell lines and primary CD4(+) T cells using single-cycle and replication-competent HIV-1 infection assays. We observed that short hairpin RNA (shRNA)-mediated stable NonO knockdown in a CD4(+) Jurkat T cell line and primary CD4(+) T cells did not affect cell viability or proliferation, but enhanced HIV-1 infection. The enhancement of HIV-1 infection in Jurkat T cells correlated with increased viral reverse transcription and gene expression. Knockdown of NonO expression in Jurkat T cells modestly enhanced HIV-1 gag mRNA expression and Gag protein synthesis, suggesting that viral gene expression and RNA regulation are the predominantly affected events causing enhanced HIV-1 replication in NonO knockdown (KD) cells. Furthermore, overexpression of NonO in Jurkat T cells reduced HIV-1 single-cycle infection by 41% compared to control cells. Our data suggest that NonO negatively regulates HIV-1 infection in CD4(+) T cells, albeit it has modest effects on early and late stages of the viral life cycle, highlighting the importance of host proteins associated with HIV-1 PIC in regulating viral replication.

  3. Is CD36 gene polymorphism in region encoding lipid-binding domain associated with early onset CAD?

    PubMed

    Rać, Monika; Safranow, Krzysztof; Kurzawski, Grzegorz; Krzystolik, Andrzej; Chlubek, Dariusz

    2013-11-01

    CD36 is a fatty acid translocase in striated muscle cells and cardiomyocytes. Some study suggested that alterations in CD36 gene may be associated with coronary artery disease (CAD) risk. The aim of the current study was to compare the frequency of CD36 variants in region encoding lipid-binding domain in Caucasian patients with early-onset CAD, no-CAD adult controls and neonates. The study group comprised 100 patients with early onset CAD. The genetic control groups were 306 infants and 40 no-CAD adults aged over 70years. Exons 4, 5 and 6 including fragments of flanking introns were studied using the denaturing high-performance liquid chromatography technique and direct sequencing. Changes detected in analyzed fragment of CD36: IVS3-6 T/C (rs3173798), IVS4-10 G/A (rs3211892), C311T (Thr104Ile, not described so far) in exon 5, G550A (Asp184Asn, rs138897347), C572T (Pro191Leu, rs143150225), G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676) in exon 6. No significant differences in the CD36 genotype, allele and haplotype frequencies were found between the three groups. Only borderline differences (p=0.066) were found between early onset CAD patients and newborns in the frequencies of 591T allele (2.00% vs 0.50%) and CGCGCGT haplotype (2.00% vs 0.50%) with both IVS3-6C and 591T variant alleles. In conclusion, CD36 variants: rs3173798, rs3211892, rs138897347, rs5956, rs143150225 rs141680676 and C311T do not seem to be involved in the risk of early-onset CAD in Caucasian population.

  4. Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site

    PubMed Central

    West, Anthony P; Schamber, Michael; Gazumyan, Anna; Golijanin, Jovana; Seaman, Michael S; Fätkenheuer, Gerd; Klein, Florian; Nussenzweig, Michel C; Bjorkman, Pamela J

    2016-01-01

    HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46–derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs. PMID:27617431

  5. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription.

    PubMed Central

    Noti, J D; Reinemann, B C; Petrus, M N

    1996-01-01

    The leukocyte integrin gene, CD11c, is transcriptionally regulated and is expressed predominantly on differentiated cells of the myelomonocytic lineage. In this study we have demonstrated that the regions -72 to -63 and -132 to -104 of the CD11c promoter contain elements responsible for phorbol ester-induced differentiation of the myeloid cell line HL60. DNase I footprinting analysis revealed that these regions can bind purified Sp1, and supershift analysis with Sp1 antibody confirmed that Sp1 in HL60 nuclear extracts could bind these regions. Transfection analysis of CD11c promoter-chloramphenicol acetyltransferase constructs containing deletions of these Sp1-binding sites revealed that these sites are essential for expression of the CD11c gene in HL60 cells but not in the T-cell line Molt4 or the cervical carcinoma cell line HeLa. Moreover, cotransfection of pPacSp1 along with these CD11c promoter-chloramphenicol acetyltransferase constructs into Sp1-deficient Drosophila Schneider 2 cells verified that these sites are essential for Sp1-dependent expression of the CD11c promoter. In vivo genomic footprinting revealed that Sp1 contacts the CD11c promoter within the regions -69 to -63 and -116 to -105 in phorbol 12-myristate 13-acetate-differentiated HL60 cells but not in undifferentiated HL60 cells or in Molt4 or HeLa cells. Cotransfection assays in HL60 cells revealed that Sp1 acts synergistically with Ap1 to activate CD11c. Further, both Sp1 sites are capable of cooperating with AP1. In vitro DNase I footprinting analysis with purified Sp1 and c-jun proteins showed that Sp1 binding could facilitate binding of c-jun. We propose that myeloid-specific expression of the CD11c promoter and is facilitated by cooperative interaction between the Sp1- and Ap1-binding sites. PMID:8649405

  6. A western type of bacterial gill disease

    USGS Publications Warehouse

    Fish, F.F.

    1935-01-01

    The first reference to a pathological condition of the gill tissues of salmonid fishes was made by Osburn in 1910. This author in describing a progressive infolding of the opercula of trout, commonly known to hatcherymen as "short gill covers," mentioned a marked proliferation on the gill epithelium as accompanying this condition. Osburn assumed that the club-like appearance of the gill filaments due to the proliferated epithelium was the result of continual irritation of the delicate gill tissue in the absence of the usual protection offered by the normal opercula. Although such a conclusion seems quite logical, it is also possible that Osburn was dealing with "short gill covers" complicated by the unknown bacterial gill disease which was subsequently described by Davis.

  7. Lignasan for bacterial gill disease

    USGS Publications Warehouse

    Rucker, Robert R.; B.J., Earp; Burrows, Roger E.

    1956-01-01

    Bacterial gill disease plagues salmon and trout in many hatcheries: some infections are sporadic, but others are continual. An inexpensive, easily applied, stable, safe chemical would be highly advantageous for treatment. The use of Roccal as a 1-hour treatment for bacterial gill disease (Fish 1947) was developed at the Leavenworth (Washington) Station of the Fish and Wildlife Service in 1942 and was quite successful. Since then, Roccal has been used extensively; but because of variability in composition, its efficacy is not consistent (Rucker et al. 1949). The objection to the variability of Roccal was overcome by using another compound, pyridylmercuric acetate, which was suggested by Van Horn and Katz (1946) as having some therapeutic therapy. Pyridylmercuric acetate was tested experimentally at the Leavenworth Station and was found to be very effective for bacterial gill disease. This compound had highly differential toxicities for bacteria and fish but was quite expensive (Rucker 1948, Burrows and Palmer 1949, Snieszko 1949). Another objection to pyridylmercuric acedate was its toxicity to rainbow trout—not to other species of trout or to salmon—at the concentration necessary to control the bacteria (Seaman 1950, Rodgers et al. 1951, Bryant 1951, Foster and Olson 1951).

  8. The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

    PubMed Central

    Li, Yali; Girardi, Enrico; Wang, Jing; Yu, Esther Dawen; Painter, Gavin F.; Kronenberg, Mitchell

    2010-01-01

    Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α–dependent structural changes in the F′ roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α–galactosyl diacylglycerol–CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes. PMID:20921281

  9. Effect of cadmium on protein synthesis in gill tissue of marine mollusc Mytilus edulis

    SciTech Connect

    Veldhuizen-Tsoerkan, M.B.; van der Mast, C.A.; Zandee, D.I. )

    1988-09-01

    Mussels have a high capacity to accumulate cadmium and other heavy metals without notable toxic effects. However, they have recently found that cadmium is toxic to M. edulis at a relatively low concentration, as anoxic survival time of the animals was significantly shortened after two weeks of exposure to 50 ppb Cd. Based on this finding, a research was started to study the toxic effects of cadmium at a macromolecular level (proteins, RNA). Mussels were exposed to 250 ppb Cd for short periods. Then excised gills were incubated with {sup 35}s-methionine for 4 hours. In the gill tissue of 7 and 15 days Cd-exposed animals, a significantly decrease in the incorporation rate of the introduced label was found of 30 and 37%, respectively. Two-dimensional gel electrophoresis was used to analyze the de novo synthesized gill tissue proteins.

  10. Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

    PubMed Central

    Rossotti, Martin A; González-Techera, Andrés; Guarnaschelli, Julio; Yim, Lucia; Camacho, Ximena; Fernández, Marcelo; Cabral, Pablo; Leizagoyen, Carmen; Chabalgoity, José A; González-Sapienza, Gualberto

    2015-01-01

    Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (˜30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera. PMID:26192995

  11. A Salmonella typhi OmpC fusion protein expressing the CD154 Trp140–Ser149 amino acid strand binds CD40 and activates a lymphoma B-cell line

    PubMed Central

    Vega, Mario I; Santos-Argumedo, Leopoldo; Huerta-Yepez, Sara; Luría-Perez, Rosendo; Ortiz-Navarrete, Vianney; Isibasi, Armado; González-Bonilla, Cesar R

    2003-01-01

    CD154 is a type II glycoprotein member of the tumour necrosis factor (TNF) ligand family, which is expressed mainly on the surface of activated T lymphocytes. The interaction with its receptor CD40, plays a central role in the control of several functions of the immune system. Structural models based on the homology of CD154 with TNF and lymphotoxin indicate that binding to CD40 involves three regions surrounding amino acids K143, R203 and Q220, and that strands W140–S149 and S198–A210 are critical for such interactions. Also, it has been reported that two recombinant CD154 fragments, including amino acid residues Y45–L261 or E108–L261 are biologically active, whereas other polypeptides, including S149–L261, are not. Therefore, we decided to construct a fusion protein inserting the W140-S149 amino acid strand (WAEKGYYTMS) in an external loop of the outer membrane protein C (OmpC) from Salmonella enterica serovar Typhi and assess its ability to bind CD40 and activate B cells. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis demonstrated that the chimeric OmpC–gp39 protein conserved its ability to form trimers. Binding to CD40 was established by three variants of enzyme-linked immunosorbent assay, a direct binding assay by coating plates with a recombinant CD40–Fc protein and through two competition assays between OmpC–gp39 and recombinant CD154 or soluble CD40–Fc. Flow cytometry analysis demonstrated that OmpC–gp39 increased the expression levels of major histocompatibility complex II, CD23, and CD80, in Raji human B-cell lymphoma similarly to an antibody against CD40. These results further support that the CD154/CD40 interaction is similar to the TNF/TNF receptor. This is the first report of a bacterial fusion protein containing a small amino acid strand form a ligand that is able to activate its cognate receptor. PMID:14511234

  12. PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205.

    PubMed

    Park, Chae Gyu; Rodriguez, Anthony; Steinman, Ralph M

    2012-10-31

    DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of the PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind to mDEC205-expressing cells. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells.

  13. Mechanism for strong binding of CdSe quantum dots to multiwall carbon nanotubes for solar energy harvesting.

    PubMed

    Azoz, Seyla; Jiang, Jie; Keskar, Gayatri; McEnally, Charles; Alkas, Alp; Ren, Fang; Marinkovic, Nebojsa; Haller, Gary L; Ismail-Beigi, Sohrab; Pfefferle, Lisa D

    2013-08-07

    As hybrid nanomaterials have myriad of applications in modern technology, different functionalization strategies are being intensely sought for preparing nanocomposites with tunable properties and structures. Multi-Walled Carbon Nanotube (MWNT)/CdSe Quantum Dot (QD) heterostructures serve as an important example for an active component of solar cells. The attachment mechanism of CdSe QDs and MWNTs is known to affect the charge transfer between them and consequently to alter the efficiency of solar cell devices. In this study, we present a novel method that enables the exchange of some of the organic capping agents on the QDs with carboxyl functionalized MWNTs upon ultrasonication. This produces a ligand-free covalent attachment of the QDs to the MWNTs. EXAFS characterization reveals direct bond formation between the CdSe QDs and the MWNTs. The amount of oleic acid exchanged is quantified by temperature-programmed decomposition; the results indicate that roughly half of the oleic acid is removed from the QDs upon functionalized MWNT addition. Additionally, we characterize the optical and structural properties of the QD-MWNT heterostructures and investigate how these properties are affected by the attachment. The steady state photoluminescence response of QDs is completely quenched. The lifetime of the PL of the QDs measured with time resolved photoluminescence shows a significant decrease after they are covalently bonded to functionalized MWNTs, suggesting a fast charge transfer between QDs and MWNTs. Our theoretical calculations are consistent with and support these experimental findings and provide microscopic models for the QD binding mechanisms.

  14. Impact of chlorpromazine self-association on its apparent binding constants with cyclodextrins: Effect of SBE(7)-beta-CD on the disposition of chlorpromazine in the rat.

    PubMed

    McIntosh, Michelle P; Leong, Nathania; Katneni, Kasiram; Morizzi, Julia; Shackleford, David M; Prankerd, Richard J

    2010-07-01

    Chlorpromazine is an antipsychotic agent with poor aqueous solubility. Complexation with SBE(7)-beta-CD can aid intravenous delivery through increasing the apparent solubility of chlorpromazine. However, chlorpromazine has also been known to self-associate. This self-association can influence its capacity to interact with other chemical species, such as cyclodextrins. This study aimed to characterise the self-association and cyclodextrin binding properties of chlorpromazine, and the effect on pharmacokinetic parameters in rats when dosed with a SBE(7)-beta-CD containing formulation. Pharmacokinetic studies of chlorpromazine in the presence and absence of SBE(7)-beta-CD were undertaken in rats. The binding constant of SBE(7)-beta-CD and chlorpromazine was studied relative to chlorpromazine concentration via fluorescence. The self-association of chlorpromazine was studied by fluorescence and UV-visible spectrophotometry. Urinary excretion of intact chlorpromazine increased in the presence of SBE(7)-beta-CD. The SBE(7)-beta-CD binding constant of chlorpromazine is highly concentration dependent and the variation can be attributed to the self-association of chlorpromazine. The apparent binding constant of chlorpromazine is highest at pharmacologically relevant concentrations, providing an explanation for the significant increase in renal chlorpromazine excretion observed in rats.

  15. Optimal lamellar arrangement in fish gills

    PubMed Central

    Park, Keunhwan; Kim, Wonjung; Kim, Ho-Young

    2014-01-01

    Fish respire through gills, which have evolved to extract aqueous oxygen. Fish gills consist of filaments with well-ordered lamellar structures, which play a role in maximizing oxygen diffusion. It is interesting that when we anatomically observe the gills of various fish species, gill interlamellar distances (d) vary little among them, despite large variations in body mass (Mb). Noting that the small channels formed by densely packed lamellae cause significant viscous resistance to water flow, we construct and test a model of oxygen transfer rate as a function of the lamellar dimensions and pumping pressure, which allows us to predict the optimal interlamellar distance that maximizes the oxygen transfer rate in the gill. Comparing our theory with biological data supports the hypothesis that fish gills have evolved to form the optimal interlamellar distances for maximizing oxygen transfer. This explains the weak scaling dependence of d on Mb: d ∼ Mb1/6. PMID:24847065

  16. Neutralization of genetically diverse HIV-1 strains by IgA antibodies to the gp120 CD4 binding site from long-term survivors of HIV infection

    PubMed Central

    Planque, Stephanie; Salas, Maria; Mitsuda, Yukie; Sienczyk, Marcin; Escobar, Miguel A.; Mooney, Jason P.; Morris, Mary-Kate; Nishiyama, Yasuhiro; Ghosh, Dipanjan; Kumar, Amit; Gao, Feng; Hanson, Carl V.; Paul, Sudhir

    2010-01-01

    Objective To identify an HIV epitope suitable for vaccine development. Design Diverse HIV-1 strains express few structurally constant regions on their surface vulnerable to neutralizing antibodies. The mostly-conserved CD4 binding site (CD4BS) of gp120 is essential for host cell binding and infection by the virus. Antibodies that recognize the CD4BS are rare, and one component of the CD4BS, the 421–433 peptide region, expresses B cell superantigenic character, a property predicted to impair the anti-CD4BS adaptive immune response. Methods IgA samples purified from the plasma of subjects with HIV infection were analyzed for the ability to bind synthetic mimetics containing the 416–433 gp120 region and full-length gp120. Infection of peripheral blood mononuclear cells by clinical HIV isolates was measured by p24 ELISA. Results IgA preparations from 3 subjects with subtype B infection for 19–21 years neutralized heterologous, coreceptor CCR5-dependent subtype A, B, C, D and AE strains with exceptional potency. The IgAs displayed specific binding of a synthetic 416–433 peptide mimetics dependent on recognition of the CD4 binding residues located in this region. Immunoadsorption, affinity chromatography and mutation procedures indicated that HIV neutralization occurred by IgA recognition of the CD4BS. Conclusions These observations identify the 421–433 peptide region as a vulnerable HIV site to which survivors of infection can produce powerful neutralizing antibodies. This indicates that the human immune system can bypass restrictions on the adaptive B cell response to the CD4BS, opening the route to targeting the 421–433 region for attaining control of HIV infection. PMID:20186035

  17. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1.

    PubMed

    Gach, Johannes S; Quendler, Heribert; Tong, Tommy; Narayan, Kristin M; Du, Sean X; Whalen, Robert G; Binley, James M; Forthal, Donald N; Poignard, Pascal; Zwick, Michael B

    2013-01-01

    Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.

  18. Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

    PubMed Central

    Mireku, S. A.; Ruetz, M.; Zhou, T.; Korkhov, V. M.; Kräutler, B.; Locher, K. P.

    2017-01-01

    BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays. PMID:28128319

  19. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    DOE PAGES

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

  20. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    SciTech Connect

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; Fouts, Timothy R.; Gallo, Robert C.; DeVico, Anthony L.; Lewis, George K.

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

  1. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions.

    PubMed

    Windheim, Mark; Southcombe, Jennifer H; Kremmer, Elisabeth; Chaplin, Lucy; Urlaub, Doris; Falk, Christine S; Claus, Maren; Mihm, Janine; Braithwaite, Myles; Dennehy, Kevin; Renz, Harald; Sester, Martina; Watzl, Carsten; Burgert, Hans-Gerhard

    2013-12-10

    The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

  2. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  3. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

  4. Heat stable antigen (mouse CD24) supports myeloid cell binding to endothelial and platelet P-selectin.

    PubMed

    Aigner, S; Ruppert, M; Hubbe, M; Sammar, M; Sthoeger, Z; Butcher, E C; Vestweber, D; Altevogt, P

    1995-10-01

    P-selectin is a Ca(2+)-dependent lectin that participates in leukocyte adhesion to vascular endothelium and platelets. Myeloid cells and a subset of T lymphocytes express carbohydrate ligands at the cell surface. Previously, we suggested that heat stable antigen (HSA/mouse CD24), an extensively glycosylated cell surface molecule on many mouse cells, is a ligand for P-selectin. Here we show that HSA mediates the binding of monocytic cells and neutrophils to P-selectin. The monocytic cell lines ESb-MP and J774, peritoneal exudate cells, and bone marrow neutrophils could bind to lipopolysaccharide-activated bend3 endothelioma cells under rotation-induced shear forces and this binding was inhibited by mAb to P-selectin and HSA. Blocking was weak at room temperature but more efficient at 4 degrees C when integrin-mediated binding was decreased. Also the adhesion of neutrophils to stimulated platelets expressing P-selectin was blocked by HSA- and P-selectin-specific mAb. Latex beads coated with purified HSA from myeloid cells bound to activated endothelioma cells or platelets, and the binding was similarly blocked by mAb to P-selectin and HSA respectively. The HSA-coated beads were stained with P-selectin-IgG, very weakly with L-selectin-IgG but not with E-selectin-IgG. The staining was dependent on divalent cations and treatment with endoglycosidase F or neuraminidase indicated that sialylated N-linked glycans were recognized. The presence of these glycans was confirmed by biosynthetic labeling studies. Our data suggest that HSA, in addition to the recently identified 160 kDa glycoprotein ligand on mouse neutrophils, belongs to a group of monospecific P-selectin ligands on myeloid cells.

  5. Structural basis for germ-line gene usage of a potent class of antibodies targeting the CD4-binding site of HIV-1 gp120.

    PubMed

    West, Anthony P; Diskin, Ron; Nussenzweig, Michel C; Bjorkman, Pamela J

    2012-07-24

    A large number of anti-HIV-1 antibodies targeting the CD4-binding site (CD4bs) on the envelope glycoprotein gp120 have recently been reported. These antibodies, typified by VRC01, are remarkable for both their breadth and their potency. Crystal structures have revealed a common mode of binding for several of these antibodies; however, the precise relationship among CD4bs antibodies remains to be defined. Here we analyze existing structural and sequence data, propose a set of signature features for potent VRC01-like (PVL) antibodies, and verify the importance of these features by mutagenesis. The signature features explain why PVL antibodies derive from a single germ-line human V(H) gene segment and why certain gp120 sequences are associated with antibody resistance. Our results bear on vaccine development and structure-based design to improve the potency and breadth of anti-CD4bs antibodies.

  6. Adaptation of human immunodeficiency virus type 1 to cells expressing a binding-deficient CD4 mutant (lysine 46 to aspartic acid).

    PubMed Central

    Choe, H R; Sodroski, J

    1995-01-01

    Human immunodeficiency virus (HIV-1) was adapted to replicate efficiently in cells expressing an altered form of the CD4 viral receptor. The mutant CD4 (46 K/D) contained a single amino acid change (lysine 46 to aspartic acid) in the CDR2 loop of domain 1, which results in a 15-fold reduction in affinity for the viral gp120 glycoprotein. The ability of the adapted virus to replicate in CD4 46 K/D-expressing cells was independently enhanced by single amino acid changes in the V2 variable loop, the V3 variable loop, and the fourth conserved (C4) region of the gp120 glycoprotein. Combinations of these amino acids in the same envelope glycoprotein resulted in additive enhancement of virus replication in cells expressing the CD4 46 K/D molecule. In cells expressing the wild-type CD4 glycoproteins, the same V2 and V3 residue changes also increased the efficiency of replication of a virus exhibiting decreased receptor-binding ability due to an amino acid change (aspartic acid 368 to glutamic acid) in the gp120 glycoprotein. In neither instance did the adaptive changes restore the binding ability of the monomeric gp120 glycoprotein or the oligomeric envelope glycoprotein complex for the mutant or wild-type CD4 glycoproteins, respectively. Thus, particular conformations of the gp120 V2 and V3 variable loops and of the C4 region allow postreceptor binding events in the membrane fusion process to occur in the context of less than optimal receptor binding. These results suggest that the fusion-related functions of the V2, V3, and C4 regions of gp120 are modulated by CD4 binding. PMID:7707502

  7. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    PubMed

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  8. Temporal pharmacokinetic/pharmacodynamic interaction between human CD3ε antigen-targeted monoclonal antibody otelixizumab and CDbinding and expression in human peripheral blood mononuclear cell static culture.

    PubMed

    Page, Kevin R; Mezzalana, Enrica; MacDonald, Alexander J; Zamuner, Stefano; De Nicolao, Giuseppe; van Maurik, Andre

    2015-11-01

    Otelixizumab is a monoclonal antibody (mAb) directed to human CD3ε, a protein forming part of the CD3/T-cell receptor (TCR) complex on T lymphocytes. This study investigated the temporal interaction between varying concentrations of otelixizumab, binding to human CD3 antigen, and expression of CD3/TCR complexes on lymphocytes in vitro, free from the confounding influence of changing lymphocyte frequencies observed in vivo. A static in vitro culture system was established in which primary human peripheral blood mononuclear cells (PBMCs) were incubated over an extended time course with titrated concentrations of otelixizumab. At each time point, free, bound, and total CD3/TCR expression on both CD4+ and CD8+ T cells and the amount of free otelixizumab antibody in the supernatant were measured. The pharmacokinetics of free otelixizumab in the culture supernatants was saturable, with a shorter apparent half-life at low concentration. Correspondingly, a rapid, otelixizumab concentration-, and time-dependent reduction in CD3/TCR expression was observed. These combined observations were consistent with the phenomenon known as target-mediated drug disposition (TMDD). A mechanistic, mathematical pharmacokinetic/pharmacodynamic (PK/PD) model was then used to characterize the free otelixizumab-CD3 expression-time relationship. CD3/TCR modulation induced by otelixizumab was found to be relatively fast compared with the re-expression rate of CD3/TCR complexes following otelixizumab removal from supernatants. In summary, the CD3/TCR receptor has been shown to have a major role in determining otelixizumab disposition. A mechanistic PK/PD model successfully captured the PK and PD in vitro data, confirming TMDD by otelixizumab.

  9. Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

    PubMed Central

    Hart, T K; Kirsh, R; Ellens, H; Sweet, R W; Lambert, D M; Petteway, S R; Leary, J; Bugelski, P J

    1991-01-01

    Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41. Images PMID:2006155

  10. Exploring the interactions and binding sites between Cd and functional groups in soil using two-dimensional correlation spectroscopy and synchrotron radiation based spectromicroscopies.

    PubMed

    Sun, Fusheng; Polizzotto, Matthew L; Guan, Dongxing; Wu, Jun; Shen, Qirong; Ran, Wei; Wang, Boren; Yu, Guanghui

    2017-03-15

    Understanding how heavy metals bind and interact in soils is essential for predicting their distributions, reactions and fates in the environment. Here we propose a novel strategy, i.e., combining two-dimensional correlation spectroscopy (2D COS) and synchrotron radiation based spectromicroscopies, for identifying heavy metal binding to functional groups in soils. The results showed that although long-term (23 yrs) organic fertilization treatment caused the accumulation of Cd (over 3 times) in soils when compared to no fertilization and chemical fertilization treatments, it significantly (p<0.05) reduced the Cd concentration in wheat grain. The 2D COS analyses demonstrated that soil functional groups controlling Cd binding were modified by fertilization treatments, providing implications for the reduced bioavailability of heavy metals in organic fertilized soils. Furthermore, correlative micro X-ray fluorescence spectromicroscopy, electron probe micro-analyzer mapping, and synchrotron-radiation-based FTIR spectromicroscopy analysis showed that Cd, minerals, and organic functional groups were heterogeneously distributed at the micro-scale in soil colloids. Only minerals, rather than organic groups, had a similar distribution pattern with Cd. Together, this strategy has a potential to explore the interactions and binding sites among heavy metals, minerals and organic components in soil.

  11. Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

    PubMed Central

    Luongo, Timothy S.; Georgiev, Ivelin S.; Matz, Julie; Schmidt, Stephen D.; Louder, Mark K.; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R.

    2013-01-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy. PMID:23843638

  12. Heavy chain-only IgG2b llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of coreceptor- and CD4-binding sites.

    PubMed

    Acharya, Priyamvada; Luongo, Timothy S; Georgiev, Ivelin S; Matz, Julie; Schmidt, Stephen D; Louder, Mark K; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R; Kwong, Peter D

    2013-09-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

  13. Identification of key residues in virulent canine distemper virus hemagglutinin that control CD150/SLAM-binding activity.

    PubMed

    Zipperle, Ljerka; Langedijk, Johannes P M; Orvell, Claes; Vandevelde, Marc; Zurbriggen, Andreas; Plattet, Philippe

    2010-09-01

    Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

  14. Changes in T cell subsets identify responders to FcR non-binding anti-CD3 mAb (teplizumab) in patients with Type 1 diabetes

    PubMed Central

    Tooley, James E; Vudattu, Nalini; Choi, Jinmyung; Cotsapas, Chris; Devine, Lesley; Raddassi, Khadir; Ehlers, Mario R; McNamara, James G; Harris, Kristina M; Kanaparthi, Sai; Phippard, Deborah; Herold, Kevan C

    2016-01-01

    The mechanisms whereby immune therapies affect progression of Type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR non-binding anti-CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8+ central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from 2 randomized clinical studies of teplizumab in patients with new and recent onset T1D. At the conclusion of therapy clinical responders showed a significant reduction in circulating CD4+ effector memory (CD4EM) T cells. Afterwards, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non-CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and non-responders vs placebo-treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D. PMID:26518356

  15. An interim report on gill disease

    USGS Publications Warehouse

    Rucker, R.R.; Johnson, H.E.; Kaydas, G.M.

    1952-01-01

    GILL DISEASE among fish, a disease which is characterized by a proliferation of the gill epithelium, has been attributed to a number of different causes. Generally, there are two recognized types: the eastern or bacterial type, in which long filamentous bacteria can always be demonstrated; and the western type, in which, by definition, bacteria cannot be demonstrated.

  16. Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

    PubMed

    Crooks, Ema T; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O'Dell, Sijy; LaBranche, Celia; Robinson, James E; Montefiori, David C; McKee, Krisha; Du, Sean X; Doria-Rose, Nicole; Kwong, Peter D; Mascola, John R; Zhu, Ping; Schief, William R; Wyatt, Richard T; Whalen, Robert G; Binley, James M

    2015-05-01

    Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

  17. Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

    PubMed Central

    Crooks, Ema T.; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S.; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O’Dell, Sijy; LaBranche, Celia; Robinson, James E.; Montefiori, David C.; McKee, Krisha; Du, Sean X.; Doria-Rose, Nicole; Kwong, Peter D.; Mascola, John R.; Zhu, Ping; Schief, William R.; Wyatt, Richard T.; Whalen, Robert G.; Binley, James M.

    2015-01-01

    Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. PMID:26023780

  18. Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

    SciTech Connect

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Ling; Robinson, Howard; Varnum, Susan M.

    2011-01-07

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR

  19. Crystal Structure of the Receptor Binding Domain of the botulinum C-D Mosiac Neurotoxin Reveals Potential Roles of Lysines 1118 and 1136 in Membrane Interactions

    SciTech Connect

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C ({approx}two-third) and BoNT/D ({approx}one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 {angstrom} resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal {beta}-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.

  20. The CD11a binding site of efalizumab in psoriatic skin tissue as analyzed by Multi-Epitope Ligand Cartography robot technology. Introduction of a novel biological drug-binding biochip assay.

    PubMed

    Bonnekoh, B; Böckelmann, R; Pommer, A J; Malykh, Y; Philipsen, L; Gollnick, H

    2007-01-01

    Efalizumab (Raptiva) is an immunomodulating recombinant humanized IgG1 monoclonal antibody that binds to CD11a, the alpha-subunit of leukocyte function antigen-1 (LFA-1). By blocking the binding of LFA-1 to ICAM-1, efalizumab inhibits the adhesion of leukocytes to other cell types and interferes with the migration of T lymphocytes to sites of inflammation (including psoriatic skin plaques). Analysis of the response in patients treated with efalizumab to date shows that distinct groups of responders and nonresponders to the drug exist. It would therefore be of great practical value to be able to predict which patients are most likely to respond to treatment, by identifying key parameters in the mechanism of action of efalizumab. Detailed investigation and detection of multiple epitopes in microcompartments of skin tissue has until recently been restricted by the available technology. However, the newly developed technique of Multi-Epitope Ligand Cartography (MELC) robot technology combines proteomics and biomathematical tools to visualize protein networks at the cellular and subcellular levels in situ, and to decipher cell functions. The MELC technique, which is outlined in this paper, was used to help characterize the binding of efalizumab to affected and unaffected psoriatic skin as compared to normal control skin under ex vivomodel conditions. Efalizumab was labeled with fluorescein isothiocyanate and integrated into a MELC library of more than 40 antibodies. These antibodies were selected for their potential to detect epitopes which may be indicative of (a) various cell types, (b) structural components of the extracellular matrix, or (c) the processes of cell proliferation, activation and adhesion. Efalizumab bound to CD11a in affected psoriatic skin by a factor 15x and 32x higher than in unaffected psoriatic skin and normal control skin, respectively. CD11a and the efalizumab binding site were primarily expressed in the extravascular dermis, whereas CD54 (ICAM

  1. Effect of cadmium on the extracellular Na⁺, K⁺, and Ca²⁺ in the gill and small intestine of goldfish Carassius auratus.

    PubMed

    Liu, Dongwu; Guo, Hong; Chen, Zhiwei; Wang, Ying

    2014-03-01

    In this study, the toxic effect of cadmium on extracellular Na(+), K(+), and Ca(2+) in the gill and small intestine of goldfish Carassius auratus was determined with the technique of ion chromatograph. Two-way ANOVA indicated that the two factors (Cd(2+) treatment and time) and the interaction factor had significant effect on the level of Na(+), K(+), and Ca(2+) in the small intestine and gill. 1.0 mg/L Cd(2+) significantly increased Ca(2+) level in the small intestine, but Ca(2+) level in the gill was significantly decreased by 1.0 and 5.0 mg/L Cd(2+) at 24, 48, and 72 h. Na(+) and K(+) level in the small intestine and gill was increased by 1.0 mg/L Cd(2+) at three time points, but increased by 5.0 mg/L Cd(2+) at a certain different time. In addition, Na(+) level was significantly decreased by 5.0 mg/L Cd(2+) at 24 or 48 h in the small intestine and gill. The results indicated that Cd(2+) played an important role in regulating the level of Na(+), K(+), and Ca(2+) in the small intestine and gill of goldfish C. auratus. A method was constructed to investigate the extracellular Na(+), K(+) and Ca(2+) in the tissues of gold fish with ion chromatography.

  2. The N276 Glycosylation Site Is Required for HIV-1 Neutralization by the CD4 Binding Site Specific HJ16 Monoclonal Antibody

    PubMed Central

    Balla-Jhagjhoorsingh, Sunita S.; Corti, Davide; Heyndrickx, Leo; Willems, Elisabeth; Vereecken, Katleen; Davis, David; Vanham, Guido

    2013-01-01

    Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb. PMID:23874792

  3. T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax).

    PubMed

    Nuñez Ortiz, N; Gerdol, M; Stocchi, V; Marozzi, C; Randelli, E; Bernini, C; Buonocore, F; Picchietti, S; Papeschi, C; Sood, N; Pallavicini, A; Scapigliati, G

    2014-12-01

    The gills of fish are a mucosal tissue that contains T cells involved in the recognition of non-self and pathogens, and in this work we describe some features of gill-associated T cells of European sea bass, a marine model species. A whole transcriptome was obtained by deep sequencing of RNA from unstimulated gills that has been analyzed for the presence of T cell-related transcripts. Of the putative expressed sequences identified in the transcriptome, around 30 were related to main functions related to T cells including Th1/Th2/Th17/Treg cell subpopulations, thus suggesting their possible presence in the branchial epithelium. The number of T cells in the gills of sea bass, measured with the specific T cell mAb DLT15 range from 10% to 20%, and IHC analysis shows their abundance and distribution in the epithelium. Leukocytes from gills are able to proliferate in the presence of lectins ConA and PHA, as measured by flow cytometry using CFSE fluorescence incorporation, and during proliferation the number of T cells counted by immunofluorescence increased. In lectin-proliferating cells the expression of T cell-related genes TRβ, TRγ, CD4, CD8α, CD45 and IL-10 increased dramatically. Our data represent a first analysis on T cell genes and on basic T cell activities of fish gills, and suggest the presence of functionally active subpopulations of T lymphocytes in this tissue.

  4. Cadmium-binding proteins from blue crabs (Callinectes sapidus) environmentally exposed to cadmium

    SciTech Connect

    Wiedow, M.A.; Kneip, T.J.; Garte, S.J.

    1982-06-01

    Two heat-stable (90/sup 0/C) cadmium-binding proteins were isolated from the hepatopancreas of Hudson River blue crabs (Callinectes sapidus) by Sephadex G-75 gel filtration chromatography. These proteins have molecular weights of 10,600 and 9,400, and ultraviolet absorbance ratios at 250/280 nm of 12.4 and 5.4, respectively. Repeated freezing and thawing and prolonged (3-6 weeks) storage resulted in protein degradation or loss of Cd-binding activity. These proteins were induced by laboratory injection of CdCl/sub 2/ in blue crabs from pristine (Chesapeake Bay) areas; however, injection of CdCl/sub 2/ into Hudson River animals yielded anomalous chromatography profiles. Cadmium-binding proteins were also identified in blue crab thoracic muscle and gill. The possibility is discussed that these proteins are a type of metallothionein and could contribute to the human toxicity of this cadmium-contaminated edible crustacean.

  5. Transcriptomic responses in rainbow trout gills upon infection with viral hemorrhagic septicemia virus (VHSV).

    PubMed

    Aquilino, Carolina; Castro, Rosario; Fischer, Uwe; Tafalla, Carolina

    2014-05-01

    It has been previously demonstrated that even though the fin bases constitute the main portal of entry of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss), an important number of chemokine genes are up-regulated in the gills upon bath exposure to the virus. Because chemokines mediate the recruitment of leukocytes through the action of specific chemokine receptors, in the current study, we have studied the transcription of several immune genes in response to a VHSV bath infection in the gills, focusing both on chemokine receptor genes and on genes characteristic of distinct leukocyte populations such as IgM, IgD, IgT, CD4, CD8, perforin and MHC-II. We have studied the response to the virus in naïve fish as well as in fish that had been previously intramuscularly (i.m.) injected with a VHSV DNA vaccine. Additionally, we have sorted both IgM(+) and CD8(+) cells from the gills of naïve and infected animals to study some of these up-regulated genes in specific leukocyte populations. Our results indicate that despite the low replication level, VHSV provokes an up-regulation of IgM, IgT, CD3 and perforin transcription together with the up-regulation of CCR7, CCR9, CXCR3B and CXCR4 mRNA levels. Interestingly, MHC-II mRNA was up-regulated and CCR7 was down-modulated in IgM(+) cells from infected gills, whereas perforin, CCR7 and CXCR4 mRNA levels were higher in sorted CD8(+) cells from infected animals. Surprisingly, when fish had been previously injected with either the empty plasmid or the VHSV DNA vaccine, these up-regulations in immune gene transcription were no longer observed. Our results point to the gills as an important site for innate and acquired viral defense.

  6. Why mushrooms form gills: efficiency of the lamellate morphology.

    PubMed

    Fischer, Mark W F; Money, Nicholas P

    2010-01-01

    Gilled mushrooms are produced by multiple orders within the Agaricomycetes. Some species form a single array of unbranched radial gills beneath their caps, many others produce multiple files of lamellulae between the primary gills, and branched gills are also common. In this largely theoretical study we modeled the effects of different gill arrangements on the total surface area for spore production. Relative to spore production over a flat surface, gills achieve a maximum 20-fold increase in surface area. The branching of gills produces the same increase in surface area as the formation of free-standing lamellulae (short gills). The addition of lamellulae between every second gill would offer a slightly greater increase in surface area in comparison to the addition of lamellulae between every pair of opposing gills, but this morphology does not appear in nature. Analysis of photographs of mushrooms demonstrates an excellent match between natural gill arrangements and configurations predicted by our model.

  7. Gilles de la Tourette syndrome.

    PubMed

    Robertson, Mary M; Eapen, Valsamma; Singer, Harvey S; Martino, Davide; Scharf, Jeremiah M; Paschou, Peristera; Roessner, Veit; Woods, Douglas W; Hariz, Marwan; Mathews, Carol A; Črnčec, Rudi; Leckman, James F

    2017-02-02

    Gilles de la Tourette syndrome (GTS) is a childhood-onset neurodevelopmental disorder that is characterized by several motor and phonic tics. Tics usually develop before 10 years of age, exhibit a waxing and waning course and typically improve with increasing age. A prevalence of approximately 1% is estimated in children and adolescents. The condition can result in considerable social stigma and poor quality of life, especially when tics are severe (for example, with coprolalia (swearing tics) and self-injurious behaviours) or when GTS is accompanied by attention-deficit/hyperactivity disorder, obsessive-compulsive disorder or another neuropsychiatric disorder. The aetiology is complex and multifactorial. GTS is considered to be polygenic, involving multiple common risk variants combined with rare, inherited or de novo mutations. These as well as non-genetic factors (such as perinatal events and immunological factors) are likely to contribute to the heterogeneity of the clinical phenotype, the structural and functional brain anomalies and the neural circuitry involvement. Management usually includes psychoeducation and reassurance, behavioural methods, pharmacotherapy and, rarely, functional neurosurgery. Future research that integrates clinical and neurobiological data, including neuroimaging and genetics, is expected to reveal the pathogenesis of GTS at the neural circuit level, which may lead to targeted interventions.

  8. The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation.

    PubMed

    Landi, Alessia; Timermans, Cristina Garcia; Naessens, Evelien; Vanderstraeten, Hanne; Stove, Veronique; Verhasselt, Bruno

    2016-03-01

    Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

  9. Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors.

    PubMed

    Sancho, D; Santis, A G; Alonso-Lebrero, J L; Viedma, F; Tejedor, R; Sánchez-Madrid, F

    2000-10-01

    CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

  10. Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa.

    PubMed

    Das, T K; Wilson, E K; Cutruzzolà, F; Brunori, M; Rousseau, D L

    2001-09-11

    The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.

  11. David Gill: clock maker to global astronomer

    NASA Astrophysics Data System (ADS)

    Haley, P. A.

    2014-04-01

    Reduction in the uncertainty of physical measurements underpinned many advances in solar and stellar parallax, the determination of longitude, geodesy, and the accurate mapping of the heavens using celestial photography in the late nineteenth century. A pioneer in these areas, who successfully made the transition from clock maker in Aberdeen to H.M. Astronomer at the Cape of Good Hope was David Gill (1843-1914); Sir David Gill, K.C.B. from 1900. This paper celebrates the first third of Gill's career in astronomy and geodesy up to the time he was made redundant from Dun Echt Observatory at the end of 1875. It highlights how his horological skills were applied to telescope design and also how his aspirations to become a global astronomer started. The paper is timed to coincide with Gill's centenary anniversary year - he died 24 January 1914.

  12. Impact of wall shear stress and ligand avidity on binding of anti-CD146-coated nanoparticles to murine tumor endothelium under flow.

    PubMed

    Thomann, Stefan; Baek, Sunhwa; Ryschich, Eduard

    2015-11-24

    The endothelial phenotype of tumor blood vessels differs from the liver and forms an important base for endothelium-specific targeting by antibody-coated nanoparticles. Although differences of shear stress and ligand avidity can modulate the nanoparticle binding to endothelium, these mechanisms are still poorly studied. This study analyzed the binding of antibody-coated nanoparticles to tumor and liver endothelium under controlled flow conditions and verified this binding in tumor models in vivo. Binding of anti-CD146-coated nanoparticles, but not of antibody was significantly reduced under increased wall shear stress and the degree of nanoparticle binding correlated with the avidity of the coating. The intravascular wall shear stress favors nanoparticle binding at the site of higher avidity of endothelial epitope which additionally promotes the selectivity to tumor endothelium. After intravenous application in vivo, pegylated self-coated nanoparticles showed specific binding to tumor endothelium, whereas the nanoparticle binding to the liver endothelium was very low. This study provides a rationale that selective binding of mAb-coated nanoparticles to tumor endothelium is achieved by two factors: higher expression of endothelial epitope and higher nanoparticle shearing from liver endothelium. The combination of endothelial marker targeting and the use of shear stress-controlled nanoparticle capture can be used for selective intratumoral drug delivery.

  13. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120.

    PubMed

    Matz, Julie; Kessler, Pascal; Bouchet, Jérôme; Combes, Olivier; Ramos, Oscar Henrique Pereira; Barin, Francis; Baty, Daniel; Martin, Loïc; Benichou, Serge; Chames, Patrick

    2013-01-01

    Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

  14. Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding.

    PubMed Central

    Hsu, E C; Dörig, R E; Sarangi, F; Marcil, A; Iorio, C; Richardson, C D

    1997-01-01

    CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis. PMID:9223509

  15. High-and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis.

    SciTech Connect

    Mishra, B.; Boyanov, M.; Bunker, B. A.; Kelly, S. D.; Kemner, K. M.; Fein, J. B.; Biosciences Division; Univ. of Notre Dame

    2010-08-01

    Bulk Cd adsorption isotherm experiments, thermodynamic equilibrium modeling, and Cd K edge EXAFS were used to constrain the mechanisms of proton and Cd adsorption to bacterial cells of the commonly occurring Gram-positive and Gram-negative bacteria, Bacillus subtilis and Shewanella oneidensis, respectively. Potentiometric titrations were used to characterize the functional group reactivity of the S. oneidensis cells, and we model the titration data using the same type of non-electrostatic surface complexation approach as was applied to titrations of B. subtilis suspensions by Fein et al. (2005). Similar to the results for B. subtilis, the S. oneidensis cells exhibit buffering behavior from approximately pH 3-9 that requires the presence of four distinct sites, with pK{sub a} values of 3.3 {+-} 0.2, 4.8 {+-} 0.2, 6.7 {+-} 0.4, and 9.4 {+-} 0.5, and site concentrations of 8.9({+-}2.6) x 10{sup -5}, 1.3({+-}0.2) x 10{sup -4}, 5.9({+-}3.3) x 10{sup -5}, and 1.1({+-}0.6) x 10{sup -4} moles/g bacteria (wet mass), respectively. The bulk Cd isotherm adsorption data for both species, conducted at pH 5.9 as a function of Cd concentration at a fixed biomass concentration, were best modeled by reactions with a Cd:site stoichiometry of 1:1. EXAFS data were collected for both bacterial species as a function of Cd concentration at pH 5.9 and 10 g/L bacteria. The EXAFS results show that the same types of binding sites are responsible for Cd sorption to both bacterial species at all Cd loadings tested (1-200 ppm). Carboxyl sites are responsible for the binding at intermediate Cd loadings. Phosphoryl ligands are more important than carboxyl ligands for Cd binding at high Cd loadings. For the lowest Cd loadings studied here, a sulfhydryl site was found to dominate the bound Cd budgets for both species, in addition to the carboxyl and phosphoryl sites that dominate the higher loadings. The EXAFS results suggest that both Gram-positive and Gram-negative bacterial cell walls have a low

  16. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.

    PubMed

    Shoham, Nitza G; Centola, Michael; Mansfield, Elizabeth; Hull, Keith M; Wood, Geryl; Wise, Carol A; Kastner, Daniel L

    2003-11-11

    Pyrin, the familial Mediterranean fever protein, is found in association with the cytoskeleton in myeloid/monocytic cells and modulates IL-1beta processing, NF-kappaB activation, and apoptosis. These effects are mediated in part through cognate interactions with the adaptor protein ASC, which shares an N-terminal motif with pyrin. We sought additional upstream regulators of inflammation by using pyrin as the bait in yeast two-hybrid assays. We now show that proline serine threonine phosphatase-interacting protein [PSTPIP1, or CD2-binding protein 1 (CD2BP1)], a tyrosine-phosphorylated protein involved in cytoskeletal organization, also interacts with pyrin. Recently, PSTPIP1/CD2BP1 mutations were shown to cause the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA), a dominantly inherited autoinflammatory disorder mediated predominantly by granulocytes. Endogenous PSTPIP1/CD2BP1 and pyrin are coexpressed in monocytes and granulocytes and can be coimmunoprecipitated from THP-1 cells. The B box segment of pyrin was necessary and the B box/coiled-coil segment sufficient for this interaction, whereas the SH3 and coiled-coil domains of PSTPIP1/CD2BP1 were both necessary, but neither was sufficient, for pyrin binding. The Y344F PSTPIP1/CD2BP1 mutation, which blocks tyrosine phosphorylation, was associated with a marked reduction in pyrin binding in pervanadate-treated cells. PAPA-associated A230T and E250Q PSTPIP1/CD2BP1 mutations markedly increased pyrin binding as assayed by immunoprecipitation and, relative to WT, these mutants were hyperphosphorylated when coexpressed with c-Abl kinase. Consistent with the hypothesis that these mutations exert a dominant-negative effect on the previously reported activity of pyrin, we found increased IL-1beta production by peripheral blood leukocytes from a clinically active PAPA patient with the A230T PSTPIP1/CD2BP1 mutation and in cell lines transfected with both PAPA-associated mutants.

  17. Specific localization of scallop gill epithelial calmodulin in cilia.

    PubMed

    Stommel, E W; Stephens, R E; Masure, H R; Head, J F

    1982-03-01

    Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system

  18. Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein

    PubMed Central

    1995-01-01

    We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS- sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two- step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30- fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP. PMID:7536794

  19. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    SciTech Connect

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.; Kwong, Peter D.; Morris, Lynn; Kirchhoff, F.

    2016-08-31

    ABSTRACT

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

    IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of

  20. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    PubMed Central

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

    2016-01-01

    ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site

  1. A novel regulatory pathway for autoimmune disease: binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance.

    PubMed

    Vandenbark, Arthur A; Meza-Romero, Roberto; Benedek, Gil; Andrew, Shayne; Huan, Jianya; Chou, Yuan K; Buenafe, Abigail C; Dahan, Rony; Reiter, Yoram; Mooney, Jeffery L; Offner, Halina; Burrows, Gregory G

    2013-02-01

    Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.

  2. Marine sediment and interstitial water: Effects on bioavailability of cadmium to gills of the clam Protothaca staminea

    SciTech Connect

    Hardy, J.T.; Schmidt, R.L.; Apts, C.W.

    1981-12-01

    A study was made to determine first, the kinetics of cadmium sorption on a natural marine sediment and second, the degree to which this sorption as well as interstitial water might effect bioavailability of cadmium to gills of the clam Protothaca staminea. Surface sediment from Sequim Bay, Washington was labelled with Cd 109 and total cadmium concentration determined by radioassay. Gills were added to three types of exposures: 1) control (0.45 um filtered seawater, 2) sediment interstitial water and 3) washed sediment. Prepared samples of gills were counted in a liquid scintillation counter. Results show that addition of a small quantity of washed sediment to the exposure system reduced cadmium accumulation by gills to only 17% of the control. Interstitial water had no significant effect. 1 table, 3 figures (JMT)

  3. Atomistic tight-binding theory of excitonic splitting energies in CdX(X = Se, S and Te)/ZnS core/shell nanocrystals

    NASA Astrophysics Data System (ADS)

    Sukkabot, Worasak; Pinsook, Udomsilp

    2017-01-01

    Using the atomistic tight-binding theory (TB) and a configuration interaction description (CI), we numerically compute the excitonic splitting of CdX(X = Se, S and Te)/ZnS core/shell nanocrystals with the objective to explain how types of the core materials and growth shell thickness can provide the detailed manipulation of the dark-dark (DD), dark-bright (DB) and bright-bright (BB) excitonic splitting, beneficial for the active application of quantum information. To analyze the splitting of the excitonic states, the optical band gaps, ground-state wave function overlaps and atomistic electron-hole interactions tend to be numerically demonstrated. Based on the atomistic computations, the single-particle and excitonic gaps are mainly reduced with the increasing ZnS shell thickness owing to the quantum confinement. In the range of the higher to lower energies, the order of the single-particle gaps is CdSe/ZnS, CdS/ZnS and CdTe/ZnS core/shell nanocrystals, while one of the excitonic gaps is CdS/ZnS, CdSe/ZnS and CdTe/ZnS core/shell nanocrystals because of the atomistic electron-hole interaction. The strongest electron-hole interactions are mainly observed in CdSe/ZnS core/shell nanocrystals. In addition, the computational results underline that the energies of the dark-dark (DD), dark-bright (DB) and bright-bright (BB) excitonic splitting are generally reduced with the increasing ZnS growth shell thickness as described by the trend of the electron-hole exchange interaction. The high-to-low splitting of the excitonic states is demonstrated in CdSe/ZnS, CdTe/ZnS and CdS/ZnS core/shell nanocrystals because of the fashion in the electron-hole exchange interaction and overlaps of the electron-hole wave functions. As the resulting calculations, it is expected that CdS/ZnS core/shell nanocrystals are the best candidates to be the source of entangled photons. Finally, the comprehensive information on the excitonic splitting can enable the use of suitable core

  4. Analysis of reactive oxygen species, Ca²+ , and Hsp70 in the gill and mantle of clams Ruditapes philippinarum exposed in cadmium.

    PubMed

    Liu, Dongwu; Chen, Zhiwei; Liu, Zhenxing

    2013-12-01

    In this study, the probes 2',7'-dichlorofluorescein diacetate (H2 DCF-DA) and Fluo-3 AM were used to investigate the instantaneous change of reactive oxygen species (ROS) and Ca(2+) in the gill and mantle of clams Ruditapes philippinarum exposed in 0.05 mg L(-1) Cd(2+) with the laser-scanning confocal microscopy. The results indicated that Ca(2+) level was declined in the gill and slightly increased in the mantle. The level of ROS was declined in the gill, while the oscillation of ROS level was observed in the mantle. These data revealed that Ca(2+) could stimulate mitochondrial activity and enhance the respiratory chain in the gill and mantle. In addition, the expression of Hsp70 was increased in the gill and mantle of clams exposed in 0.05 mg L(-1) Cd(2+) . The change of Ca(2+) and ROS level affected the expression of Hsp70 in the gill and mantle. An appropriate method was established to analyze the effects of Cd(2+) on ROS, Ca(2+) , and Hsp70 in the gill and mantle of clams with confocal microscopy. Both confocal microscopy and chemical fluorescent are valuable tools for measurement of time-dependent intracellular ROS and Ca(2+) signals.

  5. Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan

    PubMed Central

    Liang, Yu; Guttman, Miklos; Williams, James A.; Verkerke, Hans; Alvarado, Daniel

    2016-01-01

    ABSTRACT The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines. IMPORTANCE The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key

  6. Influence of potassium permanganate, cobalt chloride, and dietary supplement of vitamin B complex on the histopathological changes in gill epithelium of common carp exposed to cadmium

    SciTech Connect

    Das, B.K.; Kaviraj, A.

    1994-10-01

    Fry of common carp (Cyprinus carpio) were chronically exposed to 2.5 mg Cd/L alone and in combination with 1.0 mg KMnO{sub 4}/L or 2.0 mg CoCl{sub 2}/L or a dietary supplement of vitamin B complex at the rate of 26.5 mg/100 g food. Cadmium induced edema of primary and secondary gill lamellae, nuclear swelling, and necrosis and hypertrophy of epithelial cells of the secondary gill lamellae. Similar or more severe lamellar damages were observed with exposure to cadmium together with potassium permanganate and to cadmium together with cobalt chloride. Potassium permanganate alone was also found to produce severe edema of the gill lamellae. A dietary supplement of vitamin B complex reduced the cadmium-induced gill damages and resulted in a normal gill in exposed fish. 24 refs., 4 figs., 1 tab.

  7. The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions1

    PubMed Central

    DeFord-Watts, Laura M.; Tassin, Tara C.; Becker, Amy M.; Medeiros, Jennifer J.; Albanesi, Joseph P.; Love, Paul E.; Wülfing, Christoph; van Oers, Nicolai S. C.

    2010-01-01

    The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology. PMID:19542373

  8. Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)

    PubMed Central

    Kamata, T; Puzon, W; Takada, Y

    1995-01-01

    Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected]. Images Figure 2 Figure 3 Figure 5 PMID:7531439

  9. Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

    PubMed Central

    Goldblum, S E; Brann, T W; Ding, X; Pugin, J; Tobias, P S

    1994-01-01

    Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required. Images PMID:7509346

  10. The CD24/P-selectin binding pathway initiates lung arrest of human A125 adenocarcinoma cells.

    PubMed

    Friederichs, J; Zeller, Y; Hafezi-Moghadam, A; Gröne, H J; Ley, K; Altevogt, P

    2000-12-01

    Carbohydrates on tumor cells have been shown to play an important role in tumor metastasis. We demonstrated before that CD24, a Mr 35,000-60,000 mucine-type glycosylphosphatidylinositol-linked cell surface molecule, can function as ligand for P-selectin and that the sialylLex carbohydrate is essential for CD24-mediated rolling of tumor cells on P-selectin. To investigate the role of both antigens more closely, we transfected human A125 adenocarcinoma cells with CD24 and/or fucosyltransferase VII (Fuc TVII) cDNAs. Stable transfectants expressed CD24 and/or sialylLex. Biochemical analysis confirmed that in A125-CD24/FucTVII double transfectants, CD24 was modified with sialylLex. Only double transfectants showed rolling on P-selectin in vivo. When injected into mice, double transfectants arrested in the lungs, and this step was P-selectin dependent because it was strongly enhanced in lipopolysaccharide (LPS) pretreated wild-type mice but not in P-selectin knockout mice. CD24 modified by sialylLex was required on the tumor cells because the LPS-induced lung arrest was abolished by removal of CD24 from the cell surface by phosphatidylinositol-specific phospholipase C. A125-FucTVII single transfectants expressing sialylLex but not CD24 did not show P-selectin-mediated lung arrest. The sialylLex epitope is abundantly expressed on human carcinomas, and significant correlations between sialylLex expression and clinical prognosis exist. Our data suggest an important role for sialylLex-modified CD24 in the lung colonization of human tumors.

  11. Histopathology of fish. V. Gill disease

    USGS Publications Warehouse

    1957-01-01

    Possibly no single disease accounts for greater annual mortality than gill disease. Apparently endemic in many hatcheries, the disease is characterized by periodic sharp upsurges which are sometimes correlated with rising water temperatures, excessive foreign matter in the water (Wales and Evins 1937), or borderline nutritional conditions.

  12. Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21).

    PubMed

    Young, Kendra A; Chen, Xiaojiang S; Holers, V Michael; Hannan, Jonathan P

    2007-12-14

    Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.

  13. Cadmium kinetics in freshwater clams. Uptake of cadmium by the excised gill of Anodonta anatina

    SciTech Connect

    Holwerda, D.A.; de Knecht, J.A.; Hemelraad, J.; Veenhof, P.R.

    1989-03-01

    There are several, and sometimes conflicting, reports on metal interaction during bioaccumulation from a mixture of heavy metals by marine or estuarine organisms. Concerning the influence of zinc on Cd uptake, it was found in a previous study with the freshwater clam Anodonta cygnea that zinc retarded the accumulation of cadmium when present in a hundred-fold excess over the latter metal. In the only in vitro investigation known, it was shown that the uptake of cadmium by the excised gills of the seal mussel Mytilus edulis was not affected by co-exposure with other metal ions or by the presence of metabolic inhibitors. By contrast, bioaccumulation of cadmium in M. edulis was strongly reduced by co-exposure to zinc in a hundred-fold excess over cadmium. The clear effect of zinc on Cd accumulation in A. cygnea prompted the authors to investigate this phenomenon in an in vitro model. The primary aim was to detect whether the in vivo effect of zinc is caused by a direct influence on the gill epithelium or is sustained by a behavioral response of the animal. At the same time, the possible effect of some other exogenous factors on Cd uptake was examined. In addition, it was investigated whether the rate of in vitro uptake is a function of gill size.

  14. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    PubMed

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  15. Direct Binding of Fluorescent CdSe-ZnS Core shell Semiconductor Nanocrystals to Biological Macromolecules: Efficient Tool for Fluorescence Tagging of Biological Macromolecules

    NASA Astrophysics Data System (ADS)

    Mattoussi, Hedi; Anderson, George P.; Mauro, J. Matthew; Goldman, Ellen R.

    2000-03-01

    We present a study that describes a novel and direct approach of binding highly luminescent CdSe-ZnS core-shell nanocrystals to biological molecules for use as fluorescent probes in biosensing and diagnostics. We use dithiol-based groups as the surface capping agent, which provides nanocrystal dispersions with high quantum yield. The approach makes use of a recombinant protein, which binds directly to the dithiol cap, and provides addition stability of the quantum dots in water solutions. Hence stable and highly luminescent bound nanocrystal-biomolecules have been prepared. The present process provides aggregation-free solutions with a high luminescence yield. Combining the properties of the CdSe-ZnS (photochemical stability and wide range of emission wavelengths) and the simple binding approach, the resultant materials provide a sensitive and powerful tool for tagging of biological molecules. We will discuss the chemistry involved and present various characterization studies of these complex systems, such as photoluminescence spectroscopy, high-resolution microscopy. We will also discuss the use of these materials in immunoassays.

  16. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    PubMed

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

  17. Fusion of the Fc part of human IgG1 to CD14 enhances its binding to gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils.

    PubMed

    Vida, András; Bardoel, Bart; Milder, Fin; Majoros, László; Sümegi, Andrea; Bácsi, Attila; Vereb, György; van Kessel, Kok P M; van Strijp, Jos A G; Antal-Szalmás, Péter

    2012-08-30

    Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.

  18. Stokes shift and fine structure splitting in composition-tunable ZnxCd1-xSe nanocrystals: Atomistic tight-binding theory

    NASA Astrophysics Data System (ADS)

    Sukkabot, Worasak

    2017-02-01

    I report on the atomistic correlation of the structural properties and excitonic splitting of ternary alloy ZnxCd1-xSe wurtzite nanocrystals using the sp3s* empirical tight-binding method with the description of the first nearest neighbouring interaction and bowing effect. Based on a successful model, the computations are presented under various Zn compositions (x) and diameters of alloy ZnxCd1-xSe nanocrystals with the experimentally synthesized compositions and sizes. With increasing Zn contents (x), the optical band gaps and electron-hole coulomb energies are improved, while ground electron-hole wave function overlaps, electron-hole exchange energies, stokes shift and fine structure splitting are reduced. A composition-tunable emission from blue to yellow wavelength is obviously demonstrated. The optical band gaps, ground electron-hole wave function overlaps, electron-hole interactions, stokes shift and fine structure splitting are progressively decreased with the increasing diameters. Alloy ZnxCd1-xSe nanocrystal with Zn rich and large diameter is the best candidate to optimistically be used as a source of entangled photon pairs. The agreement with the experimental data is remarkable. Finally, the present systematic study on the structural properties and excitonic splitting predominantly opens a new perspective to understand the size- and composition-dependent properties of ZnxCd1-xSe nanocrystals with a comprehensive strategy to design the optoelectronic devices.

  19. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry

    SciTech Connect

    Meissner, Eric G.; Coffield, Vernon M.; Su Lishan . E-mail: lsu@med.unc.edu

    2005-06-05

    We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

  20. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry.

    PubMed

    Meissner, Eric G; Coffield, Vernon M; Su, Lishan

    2005-06-05

    We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for each in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.

  1. Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

    PubMed

    Dervillez, Xavier; Klaukien, Volker; Dürr, Ralf; Koch, Joachim; Kreutz, Alexandra; Haarmann, Thomas; Stoll, Michaela; Lee, Donghan; Carlomagno, Teresa; Schnierle, Barbara; Möbius, Kalle; Königs, Christoph; Griesinger, Christian; Dietrich, Ursula

    2010-10-01

    During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

  2. Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in T lymphocytes requires CD4-gp120 binding.

    PubMed Central

    Marcuzzi, A; Lowy, I; Weinberger, O K

    1992-01-01

    Cells expressing human immunodeficiency virus type 1 (HIV-1) tat can transactivate the HIV-1 long terminal repeat (LTR) in cocultured T lymphocytes. In this report, we describe the molecular requirements for transcellular activation of the LTR in Jurkat cells. An analysis with deletion mutants and blocking antibodies demonstrated a requirement for env expression in addition to tat expression for transcellular activation to occur. The results suggest that the transient association of CD4 and gp120 in cocultured cells is required for tat-mediated transcellular activation. The events that follow CD4-gp120 binding in transactivation, however, do not require the gp120-neutralizing domain, in contrast to HIV-mediated fusion and infection. The consequences of this interaction on cellular function are currently under investigation. Images PMID:1351104

  3. NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching.

    PubMed

    Lippert, Lisa G; Hallock, Jeffrey T; Dadosh, Tali; Diroll, Benjamin T; Murray, Christopher B; Goldman, Yale E

    2016-03-16

    We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.

  4. Prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory CD8 T cells.

    PubMed

    León, Beatriz; Ballesteros-Tato, André; Randall, Troy D; Lund, Frances E

    2014-07-28

    The commitment of naive CD8 T cells to effector or memory cell fates can occur after a single day of antigenic stimulation even though virus-derived antigens (Ags) are still presented by DCs long after acute infection is resolved. However, the effects of extended Ag presentation on CD8 T cells are undefined and the mechanisms that regulate prolonged Ag presentation are unknown. We showed that the sustained presentation of two different epitopes from influenza virus by DCs prevented the premature contraction of the primary virus-specific CD8 T cell response. Although prolonged Ag presentation did not alter the number of memory CD8 T cells that developed, it was essential for programming the capacity of these cells to proliferate, produce cytokines, and protect the host after secondary challenge. Importantly, prolonged Ag presentation by DCs was dependent on virus-specific, isotype-switched antibodies (Abs) that facilitated the capture and cross-presentation of viral Ags by FcγR-expressing DCs. Collectively, our results demonstrate that B cells and Abs can regulate the quality and functionality of a subset of antiviral CD8 T cell memory responses and do so by promoting sustained Ag presentation by DCs during the contraction phase of the primary T cell response.

  5. Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.

    PubMed

    Naarding, Marloes A; Dirac, Annette M; Ludwig, Irene S; Speijer, Dave; Lindquist, Susanne; Vestman, Eva-Lotta; Stax, Martijn J; Geijtenbeek, Teunis B H; Pollakis, Georgios; Hernell, Olle; Paxton, William A

    2006-10-01

    A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

  6. Diverse CdII coordination complexes derived from bromide isophthalic acid binding with auxiliary N-donor ligands

    NASA Astrophysics Data System (ADS)

    Tang, Meng; Dong, Bao-Xia; Wu, Yi-Chen; Yang, Fang; Liu, Wen-Long; Teng, Yun-Lei

    2016-12-01

    The coordination characteristics of 4-bromoisophthalic acid (4-Br-H2ip) have been investigated in a series of CdII-based frameworks. Hydrothermal reactions of CdII salts and 4-Br-H2ip together with flexible or semiflexible N-donor auxiliary ligands resulted in the formation of four three-dimensional coordination complexes with diverse structures: {Cd(bix)0.5(bix)0.5(4-Br-ip)]·H2O}n (1), [Cd(bbi)0.5(bbi)0.5(4-Br-ip)]n (2), {[Cd(btx)0.5(4-Br-ip)(H2O)]·0.5CH3OH·H2O}n (3) and {[Cd(bbt)0.5(4-Br-ip)(H2O)]·3·5H2O}n (4). These compounds were characterized by elemental analyses, IR spectra, single-crystal and powder X-ray diffraction. They displayed diverse structures depending on the configuration of the 4-connected metal node, the coordination mode of the 4-Br-H2ip, the coordination ability and conformationally flexibility of the N-donor auxiliary. Compound 1 exhibits 3-fold interpenetrated 66 topology and compound 2 has a 412 topology. Compounds 3-4 have similar 3D pillar-layered structures based on 3,4-connected binodal net with the Schläfli symbol of (4·38). The thermal stabilities and photoluminescence properties of them were discussed in detail.

  7. The McGill-Costa Rica Project

    PubMed Central

    Davis, M. William L.; Haggerty, Jeannie; Filion-Laporte, Liliane

    1992-01-01

    In 1987, health authorities in Costa Rica started a training program in family and community medicine and requested assistance from the Department of Family Medicine at McGill University. The authors outline the health care system in Costa Rica and the background of the project. The design, implementation, and progress of the project to date are discussed. The problems associated with this kind of project and its future are also discussed. Imagesp1152-a PMID:21221332

  8. Vascular anatomy of the fish gill.

    PubMed

    Olson, Kenneth R

    2002-08-01

    The fish gill is the most physiologically diversified vertebrate organ, and its vasculature the most intricate. Application of vascular corrosion techniques that couple high-fidelity resins, such as methyl methacrylate, with scanning electron microscopy yields three-dimensional replicas of the microcirculation that have fostered a better appreciate gill perfusion pathways. This is the focus of the present review. Three vascular networks can be identified within the gill filament. The arterioarterial (respiratory) pathway consists of the lamellae and afferent and efferent segments of the branchial and filamental arteries and lamellar arterioles. The body of the filament contains two post-lamellar pathways: the interlamellar and nutrient. The interlamellar system is an extensive ladder-like network of thin-walled, highly distensible vessels that traverses the filament between, and parallel to, the lamellae and continues around the afferent and efferent borders of the filament. Interlamellar vessels are supplied by short, narrow-bore feeder vessels from the medial wall of the efferent filamental artery. A myriad of narrow-bore, tortuous arterioles arise from the basal efferent filamental artery and efferent branchial artery and anastomose to form the nutrient circulation of the arch and filament. In the filament body, nutrient capillaries and interlamellar vessels are often closely associated, and the former may ultimately drain into the latter. Many of the anatomical characteristics of interlamellar vessels are strikingly similar to those of mammalian lymphatic capillaries, with the exception that interlamellar vessels are directly fed by arteriovenous-like anastomoses. It is likely that gill interlamellar and mammalian lymphatics are physiologically, if not embryologically, equivalent.

  9. Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides

    PubMed Central

    2016-01-01

    A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term. 1H–15N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan–lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides. PMID:26129647

  10. Proflavine binding to poly(rC-rA) inverts the CD spectrum but not the helix handedness.

    PubMed

    Westhof, E; Sundaralingam, M

    1984-08-01

    The interaction of proflavine hemisulfate with the sodium salt of poly(rC-rA) in solution (unbuffered) yields an inverted (mirror-like) circular dichroism (CD) spectrum to that of the free poly(rC-rA). Simultaneously, an induced negative Cotton effect appears in the proflavine band region with a maximum at 467 nm and a slight shoulder at 420 nm. This observation may be explained as resulting from the formation of a poly(rC-rA).proflavine complex with the polynucleotide existing as a right-handed parallel chain duplex with the proflavine intercalated between the CpA sequence and not the ApC sequence. The intercalation geometry here is expected to be analogous to that found in the crystal structure of the dinucleotide CpA.proflavine complex (Westhof et al. J. Mol. Biol., 1981) which forms a miniature right-handed helix. Although normally an inverted spectra could be attributed to a reversal in the helix handedness, the similarity in the 31P nuclear magnetic resonance spectra between the free and proflavine bound poly(rC-rA) indicates that their handedness is the same. The inverted CD spectrum may be a result of the different stacking orientation between the intercalated proflavine and the A-A base-pair on one hand and the triply hydrogen bonded protonated C-C base-pair on the other.

  11. Mapping of the C3d ligand binding site on complement receptor 2 (CR2/CD21) using nuclear magnetic resonance and chemical shift analysis.

    PubMed

    Kovacs, James M; Hannan, Jonathan P; Eisenmesser, Elan Z; Holers, V Michael

    2009-04-03

    Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, Epstein-Barr virus gp350, interferon-alpha, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC(50). C3d was titrated into (15)N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues Arg(13), Tyr(16), Arg(28), Tyr(29), Ser(32), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(101), Asn(105), and Ser(109). SCR1 and SCR2 demonstrated distinct binding modes. The CR2 binding surface incorporating SCR1 is inconsistent with a previous x-ray CR2-C3d co-crystal analysis but consistent with mutagenesis, x-ray neutron scattering, and inhibitory monoclonal antibody epitope mapping. Titration with C3dp1 yielded chemical shift changes (Arg(13), Tyr(16), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(105), and Ser(109)) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d.

  12. Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.

    PubMed

    Schröder, N W; Opitz, B; Lamping, N; Michelsen, K S; Zähringer, U; Göbel, U B; Schumann, R R

    2000-09-01

    Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.

  13. Studies on the interactions of SAP-1 (an N-terminal truncated form of cystatin S) with its binding partners by CD-spectroscopic and molecular docking methods.

    PubMed

    Yadav, Vikash Kumar; Mandal, Rahul Shubhra; Puniya, Bhanwar Lal; Singh, Sarman; Yadav, Savita

    2015-01-01

    SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of β-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.

  14. Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity

    PubMed Central

    Lavado, Ramon; Bammler, Theo K.; Gallagher, Evan P.; Stapleton, Patricia L.; Beyer, Richard P.; Farin, Federico M.; Hardiman, Gary; Schlenk, Daniel

    2015-01-01

    Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors. PMID:26260986

  15. Biomarkers of environmental stress in gills of ribbed mussel Aulacomya atra atra (Nuevo Gulf, Northern Patagonia).

    PubMed

    Giarratano, Erica; Gil, Mónica N; Malanga, Gabriela

    2014-09-01

    In this study, we assessed in gills of native ribbed mussels Aulacomya atra atra from three sites within Nuevo Gulf (Northern Patagonia) several biomarkers such as reactive oxygen species (ROS), lipid radicals (LR), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and metallothionein (MT). Furthermore, concentrations of main trace metals (Fe, Al, Zn, Cu, Cd and Pb) were quantified in mussel tissue. Results showed significant induction of SOD, GST, MT and MDA, as well as, higher concentration of Fe, Al and Cd in winter than in summer. The high MDA content measured in mussels from Folías Wreck seemed to be caused by the very high levels of Fe that would come from the corrosion of the vessel. Mussels from the control site Punta Cuevas presented the lowest levels of Cd and the highest of Al in winter. Despite positive correlations were found between Al and GST and MT, no spatial differentiation was detected in those biomarkers. On the other hand, MT was only related to Al been most likely influenced by environmental variables than by the trace metals. It has to be highlighted that the relationship detected among water temperature, nutrients and antioxidant responses in gills is probably related to the fact that this tissue is in direct contact with water and it is sensitive to its fluctuations. Taking into account that mussel gill is a tissue actively proliferating and the first target of contaminants present in water, so that changes in its antioxidant system can provide an earlier warning signal than in other tissues.

  16. Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

    SciTech Connect

    Corey, Elizabeth A.; Iorio, Ronald M.

    2009-01-05

    Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation of fusion by these two paramyxoviruses.

  17. Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin, ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

    PubMed Central

    Barkham, Timothy M. S.; Tang, Wenying; Kemeny, David M.; Chee, Cynthia Bin-Eng; Wang, Yee T.

    2014-01-01

    The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154+ TNF-α+ cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154+ TNF-α+ IFN-γ+ IL-2+ and CD154+ TNF-α+ CXCR3+. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB. PMID:25520147

  18. Structural insights into the functional role of the Hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype C/D.

    PubMed

    Zhang, Yanfeng; Gardberg, Anna S; Edwards, Thomas E; Sankaran, Banumathi; Robinson, Howard; Varnum, Susan M; Buchko, Garry W

    2013-07-01

    Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a β-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T

  19. Neisserial outer membrane vesicles bind the coinhibitory receptor carcinoembryonic antigen-related cellular adhesion molecule 1 and suppress CD4+ T lymphocyte function.

    PubMed

    Lee, Hannah S W; Boulton, Ian C; Reddin, Karen; Wong, Henry; Halliwell, Denise; Mandelboim, Ofer; Gorringe, Andrew R; Gray-Owen, Scott D

    2007-09-01

    Pathogenic Neisseria bacteria naturally liberate outer membrane "blebs," which are presumed to contribute to pathology, and the detergent-extracted outer membrane vesicles (OMVs) from Neisseria meningitidis are currently employed as meningococcal vaccines in humans. While the composition of these vesicles reflects the bacteria from which they are derived, the functions of many of their constituent proteins remain unexplored. The neisserial colony opacity-associated Opa proteins function as adhesins, the majority of which mediate bacterial attachment to human carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). Herein, we demonstrate that the Opa proteins within OMV preparations retain the capacity to bind the immunoreceptor tyrosine-based inhibitory motif-containing coinhibitory receptor CEACAM1. When CD4(+) T lymphocytes were exposed to OMVs from Opa-expressing bacteria, their activation and proliferation in response to a variety of stimuli were effectively halted. This potent immunosuppressive effect suggests that localized infection will generate a "zone of inhibition" resulting from the diffusion of membrane blebs into the surrounding tissues. Moreover, it demonstrates that OMV-based vaccines must be developed from strains that lack CEACAM1-binding Opa variants.

  20. Atomistic tight-binding computations in structural and optical properties of CdSe/ZnSe/ZnS core/multi-shell nanocrystals

    NASA Astrophysics Data System (ADS)

    Sukkabot, Worasak

    2016-07-01

    In the present paper, I attempt to theoretically describe, analyze and compare the structural and optical properties in the core/multi-shell nanocrystal structure of a cadmium selenide (CdSe) core surrounded by zinc selenide (ZnSe) inner and zinc sulphide (ZnS) external growth shells. The atomistic tight-binding model (TB) and a configuration interaction method (CI) are implemented to calculate the single-particle spectra, optical band gaps, ground-state wave function overlaps, ground-state oscillation strengths, ground-state coulomb energies, ground-state exchange energies and Stokes shift as a function of ZnS external growth shell thicknesses. I underline that these computations are principally sensitive with the ZnS external growth shell thickness. The reduction of the optical band gaps, overlaps of ground electron-hole wave function, electron-hole interactions and Stokes shift is realized with the increasing ZnS external growth shell thickness. The improvement of the optical intensities is mainly achieved by including the ZnS exterior growth shell encapsulation. Importantly, the optical band gaps based on atomistic tight-binding theory are in a good agreement with the experiment. Finally, this emphasizes that the external passivation shell can now be engineered in a defined way, thus leading to manipulate the natural behaviors of nanodevices based on the scrutinized core/multi-shell nanocrystals.

  1. Gilles de la Tourette's syndrome in monozygotic twins

    PubMed Central

    Waserman, Jack; Lal, Samarthji; Gauthier, Serge

    1983-01-01

    Concordance is reported of Gilles de la Tourette syndrome in a male twin pair in whom phenotyping revealed a >98·7% probability that they were monozygotic. The development and extent of the illness differed markedly in the two subjects. Our findings are compatible with the view that there is a genetic form of Gilles de la Tourette syndrome. PMID:6573436

  2. Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d.

    PubMed

    Hannan, Jonathan P; Young, Kendra A; Guthridge, Joel M; Asokan, Rengasamy; Szakonyi, Gerda; Chen, Xiaojiang S; Holers, V Michael

    2005-02-25

    We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.

  3. Monoclonal Antibodies to V2, V3, the CD4-binding site and gp41 HIV-1 Mediate Phagocytosis in a Dose-dependent Manner.

    PubMed

    Musich, Thomas; Li, Liuzhe; Liu, Lily; Zolla-Pazner, Susan; Robert-Guroff, Marjorie; Gorny, Miroslaw K

    2017-01-25

    In light of weak or absent neutralizing activity mediated by anti-V2 monoclonal Abs (mAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP) which is an important element of anti-HIV-1 immunity. We tested six anti-V2 mAbs and compared them with 21 mAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from HIV-1 chronically infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664 or BG505 DS-SOSIP.664 complexed with mAbs. The ADCP activity measured by the area under the curve showed significantly higher activity of anti-gp41 mAbs compared to three other groups of mAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 mAbs were dominant over anti-V3 and anti-CD4bs against clade C gp120ZM109. ADCP mediated by V2 and V3 mAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting a closed envelope conformation better exposes the variable loops. Two IgG3 mAbs against V2 and V3 regions displayed dominant ADCP activity over a panel of IgG1 mAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 mAbs were compared to IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers with some higher activity of anti-V2 mAbs over anti-V3 and anti-CD4bs mAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.

  4. Influence of chemical and environmental stresses on metal-binding proteins: Species-dependent effects

    SciTech Connect

    Baer, K.N.

    1988-01-01

    The development of tolerance to cadmium toxicity was investigated in mammals. In adult mice pretreated with 20 mg Cd/kg, no mortality was observed following administration of a 100 mg/kg cadmium challenge dose. In animals receiving prior exposure to cold stress a mortality of 40% was observed, while in animals receiving no pretreatment an 80% mortality was observed following cadmium challenge. Analysis of the metal-binding proteins using G-75 gel-filtration chromatography revealed that MT-like protein was responsible, in part, for the observed tolerance to cadmium toxicity. For example, following 20 mg Cd/kg and cold pretreatment, the MT-like reserve capacity was 56 and 42 nmoles cadmium, respectively, compared to a control value of 12 nmoles cadmium. The influence of pretreatments on the subcellular distribution of cadmium was also examined. The influence of chemical and environmental stresses on metal-binding proteins in teleosts was investigated. Following cadmium exposure, cadmium increased in the MT fraction in both the gill and liver. However, following exposure to environmental stresses such as cold and hypoxia, significant decreases in zinc and copper were observed in the gill MT fraction, as compared to control. In the liver, no significant alterations were observed in the MT fraction, as compared to control.

  5. Purification and characterization studies of cadmium-binding proteins from the American oyster, Crassostrea virginica

    SciTech Connect

    Fowler, B.A.; Engel, D.W.; Brouwer, M.

    1986-03-01

    The previously reported low molecular weight cadmium-binding protein (CdBP) from the American oyster, Crassostrea virginica, has been further purified and characterized by improved technical methods. The internal organ distribution of the protein within the oyster and effects of life cycle/season on CdBP production also have been evaluated. CdBP isolated by extended ion-exchange gradients or double ion-exchange chromatography followed by HPLC analysis possesses an electrophoretic R/sub f/ of about 0.7 and contains relatively little Zn. Cysteine, lysine, and glycine are the dominant amino acids. When ion-exchange columns are developed with NaCl gradients, the aromatic residues tryptophan, tyrosine, and phenylalanine are found to be present. The ultraviolet absorption spectrum of CdBP also was variable, with 250/280 nm ratios ranging from 17:1 immediately after ion-exchange chromatography to 2:1 following concentration procedures. Internal organ distribution studies showed that the visceral mass contained most of the Cd present with lesser amounts in the gills and mantle. Relative oyster dormancy during the winter also reduces CdBP production in response to Cd, and the protein is obtained most readily during the fall and spring.

  6. The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®.

    PubMed

    Ganguly, Bishu; Balasa, Balaji; Efros, Lyubov; Hinton, Paul R; Hartman, Stephen; Thakur, Archana; Xiong, Joanna M; Schmidt, Brian; Robinson, Randy R; Sornasse, Thierry; Vexler, Vladimir; Sheridan, James P

    2016-10-01

    The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax.

  7. Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex*

    PubMed Central

    Ranoa, Diana Rose E.; Kelley, Stacy L.; Tapping, Richard I.

    2013-01-01

    Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins. PMID:23430250

  8. Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula.

    PubMed

    Park, Jin-Sung; Chung, Soohee; Park, Il-Seon; Kim, Yangsun; Koh, Chul- Hwan; Lee, In-Sook

    2002-04-01

    Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.

  9. Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind C1q.

    PubMed

    McGreal, Eamon P; Ikewaki, Nobunao; Akatsu, Hiroyasu; Morgan, B Paul; Gasque, Philippe

    2002-05-15

    It has been suggested that the human C1qRp is a receptor for the complement component C1q; however, there is no direct evidence for an interaction between C1q and C1qRp. In this study, we demonstrate that C1q does not show enhanced binding to C1qRp-transfected cells compared with control cells. Furthermore, a soluble recombinant C1qRp-Fc chimera failed to interact with immobilized C1q. The proposed role of C1qRp in the phagocytic response in vivo is also unsupported in that we demonstrate that this molecule is not expressed by macrophages in a variety of human tissues and the predominant site of expression is on endothelial cells. Studies on the rodent homolog of C1qRp, known as AA4, have suggested that this molecule may function as an intercellular adhesion molecule. Here we show that C1qRp is the Ag recognized by several previously described mAbs, mNI-11 and two anti-CD93 Abs (clones X2 and VIMD2b). Interestingly, mNI-11 (Fab') has been shown to promote monocyte-monocyte and monocyte-endothelial cell adhesive interactions. We produced a recombinant C1qRp-Fc chimera containing the C-type lectin-like domain of C1qRp and found specific binding to vascular endothelial cells in sections of inflamed human tonsil, indicating the presence of a C1qRp ligand at this site. This interaction was Ca(2+) independent and was not blocked by our anti-C1qRp mAb BIIG-4, but was blocked by the proadhesive mAb mNI-11. Collectively, these data indicate that C1qRp is not a receptor for C1q, and they support the emerging role of C1qRp (here renamed CD93) in functions relevant to intercellular adhesion.

  10. Gill morphometrics of the thresher sharks (Genus Alopias): Correlation of gill dimensions with aerobic demand and environmental oxygen.

    PubMed

    Wootton, Thomas P; Sepulveda, Chugey A; Wegner, Nicholas C

    2015-05-01

    Gill morphometrics of the three thresher shark species (genus Alopias) were determined to examine how metabolism and habitat correlate with respiratory specialization for increased gas exchange. Thresher sharks have large gill surface areas, short water-blood barrier distances, and thin lamellae. Their large gill areas are derived from long total filament lengths and large lamellae, a morphometric configuration documented for other active elasmobranchs (i.e., lamnid sharks, Lamnidae) that augments respiratory surface area while limiting increases in branchial resistance to ventilatory flow. The bigeye thresher, Alopias superciliosus, which can experience prolonged exposure to hypoxia during diel vertical migrations, has the largest gill surface area documented for any elasmobranch species studied to date. The pelagic thresher shark, A. pelagicus, a warm-water epi-pelagic species, has a gill surface area comparable to that of the common thresher shark, A. vulpinus, despite the latter's expected higher aerobic requirements associated with regional endothermy. In addition, A. vulpinus has a significantly longer water-blood barrier distance than A. pelagicus and A. superciliosus, which likely reflects its cold, well-oxygenated habitat relative to the two other Alopias species. In fast-swimming fishes (such as A. vulpinus and A. pelagicus) cranial streamlining may impose morphological constraints on gill size. However, such constraints may be relaxed in hypoxia-dwelling species (such as A. superciliosus) that are likely less dependent on streamlining and can therefore accommodate larger branchial chambers and gills.

  11. Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

    PubMed Central

    O'Rourke, Sara M.; Schweighardt, Becky; Phung, Pham; Mesa, Kathryn A.; Vollrath, Aaron L.; Tatsuno, Gwen P.; To, Briana; Sinangil, Faruk; Limoli, Kay; Wrin, Terri

    2012-01-01

    The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals. PMID:22933284

  12. Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2*♦

    PubMed Central

    Kuronuma, Koji; Mitsuzawa, Hiroaki; Takeda, Katsuyuki; Nishitani, Chiaki; Chan, Edward D.; Kuroki, Yoshio; Nakamura, Mari; Voelker, Dennis R.

    2009-01-01

    Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-α production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoyl-phosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important anti-inflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung. PMID:19584052

  13. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    PubMed Central

    Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall’Acqua, William; Damschroder, Melissa; Hammond, Scott A.

    2012-01-01

    MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. PMID:22574157

  14. CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

    PubMed

    Zhang, Chunyan; Xin, Hong; Zhang, Wang; Yazaki, Paul J; Zhang, Zhifang; Le, Keith; Li, Wenzhao; Lee, Heehyoung; Kwak, Larry; Forman, Stephen; Jove, Richard; Yu, Hua

    2016-04-19

    The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

  15. Concentrations of metals and potential metal-binding compounds and speciation of Cd, Zn and Cu in phloem and xylem saps from castor bean plants (Ricinus communis) treated with four levels of cadmium.

    PubMed

    Hazama, Kenji; Nagata, Shinji; Fujimori, Tamaki; Yanagisawa, Shuichi; Yoneyama, Tadakatsu

    2015-06-01

    We examined the concentrations of metals (Cd, Zn, Cu, Fe and Mn) and potential metal-binding compounds [nicotianamine (NA), thiol compounds and citrate] in xylem and phloem saps from 4-week-old castor bean plants (Ricinus communis) treated with 0 (control), 0.1, 1.0, and 10 μM Cd for 3 weeks. Treatment with 0.1 and 1 μM Cd produced no visible damage, while 10 μM Cd retarded growth. Cadmium concentrations in both saps were higher than those in the culture solution at 0.1 μM, similar at 1.0 μM and lower at 10 μM. Cd at 10 μM reduced Cu and Fe concentrations in both saps. NA concentrations measured by capillary electrophoresis-mass spectrometry (MS) in xylem sap (20 μM) were higher than the Cu concentrations, and those in phloem sap (150 μM) were higher than those of Zn, Fe and Cu combined. Reduced glutathione concentrations differed in xylem and phloem saps (1-2 and 30-150 μM, respectively), but oxidized glutathione concentrations were similar. Phloem sap phytochelatin 2 concentration increased from 0.8 μM in controls to 8 μM in 10 μM Cd. Free citrate was 2-4 μM in xylem sap and 70-100 μM in phloem sap. Total bound forms of Cd in phloem and xylem saps from 1 μM Cd-treated plants were 54 and 8%, respectively. Treatment of phloem sap with proteinaseK reduced high-molecular compounds while increasing fractions of low-molecular Cd-thiol complexes. Zinc-NA, Fe-NA and Cu-NA were identified in the phloem sap fraction of control plants by electrospray ionization time-of-flight MS, and the xylem sap contained Cu-NA.

  16. Gill morphometry of the red drum, Sciaenops ocellatus.

    PubMed

    Don Stevens, E

    1992-08-01

    The structure and morphometry of the gills of the marine teleost, red drum, have been studied. The present analysis of gas exchange area of fish gills is one of the most intensive and the results are compared to less intense averaging methods. Based on the gill area estimates, red drum falls into the category of a fish of intermediate activity. Its gill clearly has an exchange area less than that of the tunas, but is slightly greater than that of trout or bass. The three components that contribute to total exchange area (filament length, lamellar density, and area of individual lamellae) are not all greater in species with a greater total exchange area. The best correlate is total filament length.

  17. McGill's Integrated Civil and Common Law Program.

    ERIC Educational Resources Information Center

    Morissette, Yves-Marie

    2002-01-01

    Describes the bijural program of McGill University Faculty of Law. The program educates all first-degree law students in both the common law and civil law traditions, preparing them for the increasing globalization of legal practice. (EV)

  18. Insights into the nature of uranium target proteins within zebrafish gills after chronic and acute waterborne exposures.

    PubMed

    Bucher, Guillaume; Mounicou, Sandra; Simon, Olivier; Floriani, Magali; Lobinski, Ryszard; Frelon, Sandrine

    2016-03-01

    New data on the nature of the protein targets of uranium (U) within zebrafish gills were collected after waterborne exposure, with the aim of a better understanding of U toxicity mechanisms. Some common characteristics of the U protein target binding properties were found, such as their role in the regulation of other essential metals and their phosphorus content. In total, 21 potential protein targets, including hemoglobin, are identified and discussed in terms of the literature.

  19. A CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis.

    PubMed

    Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Guerra, Laura; Picchietti, Simona; Costantini, Susan; Facchiano, Angelo M; Zou, Jun; Secombes, Chris J; Scapigliati, Giuseppe

    2008-06-01

    CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell co-receptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4h and a decrease after 24h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.

  20. Purification and characterization studies of cadmium-binding proteins from the American oyster, Crassostrea virginica.

    PubMed

    Fowler, B A; Engel, D W; Brouwer, M

    1986-03-01

    The previously reported low molecular weight cadmium-binding protein (CdBP) from the American oyster, Crassostrea virginica, has been further purified and characterized by improved technical methods. The internal organ distribution of the protein within the oyster and effects of life cycle/season on CdBP production also have been evaluated. CdBP isolated by extended ion-exchange gradients or double ion-exchange chromatography followed by HPLC analysis possesses an electrophoretic Rf of about 0.7 and contains relatively little Zn, as previously reported. Cysteine, lysine, and glycine are the dominant amino acids. When ion-exchange columns are developed with NaCl gradients, the aromatic residues tryptophan, tyrosine, and phenylalanine are found to be present, but these may be largely removed depending upon whether the protein is denatured and carboxymethylated prior to analysis. The ultraviolet absorption spectrum of CdBP also was variable, with 250/280 nm ratios ranging from 17:1 immediately after ion-exchange chromatography to 2:1 following concentration procedures. Internal organ distribution studies showed that the visceral mass contained most of the Cd present with lesser amounts in the gills and mantle. In contrast with mammals, CdBP accounts for only about 30% of the total cell Cd burden in these tissues. Cu displacement of Cd from the protein is a particular problem during the summer spawning season and appears to stem from altered Cu metabolism during this period. Relative oyster dormancy during the winter also reduces CdBP production in response to Cd, and the protein is obtained most readily during the fall and spring.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. CCR7 is mainly expressed in teleost gills, where it defines an IgD+IgM- B lymphocyte subset.

    PubMed

    Castro, Rosario; Bromage, Erin; Abós, Beatriz; Pignatelli, Jaime; González Granja, Aitor; Luque, Alfonso; Tafalla, Carolina

    2014-02-01

    Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7(+) cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7(+) cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7(+) cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD(+) cells in the gills expressed CCR7. Intriguingly, the IgD(+)CCR7(+) population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7(+) cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD(+)memIgM(-) B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD(+)IgM(-) subsets in mammals are discussed.

  2. Structural Insights into the Functional Role of the Hcn Sub-domain of the Receptor-Binding Domain of the Botulinum Neurotoxin Mosaic Serotype C/D

    SciTech Connect

    Zhang, Yanfeng; Gardberg, Anna; Edwards, Tom E.; Sankaran, Banumathi; Robinson, Howard; Varnum, Susan M.; Buchko, Garry W.

    2013-07-01

    Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding module (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell’s membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a B-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been indentified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) molecule bound in an hydrophobic cleft between B-strands in the B-sheet jelly fold roll of the Hcn sub-domain. The molecule is completely engulfed in the cleft, making numerous hydrophobic (Y932, S959, W966, and D1042) and hydrophilic (S935, W977, L979, N1013, and I1066) contacts with the protein’s side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid

  3. Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

    PubMed

    Valdenegro-Vega, Victoria A; Crosbie, Phil; Bridle, Andrew; Leef, Melanie; Wilson, Richard; Nowak, Barbara F

    2014-09-01

    The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

  4. Effects of exposure to multiple trace metals on biochemical, histological and ultrastructural features of gills of a freshwater fish, Channa punctata Bloch.

    PubMed

    Pandey, Suwarna; Parvez, Suhel; Ansari, Rizwan Ahamd; Ali, Mehboob; Kaur, Manpreet; Hayat, Faisal; Ahmad, Firoz; Raisuddin, Sheikh

    2008-08-11

    The trace metals are frequently encountered as mixtures of essential and non-essential elements. Therefore, evaluation of their toxic effects individually does not offer a realistic estimate of their impact on biological processes. We studied effects of a mixture of four essential and toxic metals (Cu, Cd, Fe and Ni) on biochemical and morphological characteristics of the gills of a biomarker freshwater fish Channa punctata (Bloch) using environmentally relevant concentrations. Fish were exposed to metal mixture through tank water for 7, 15 and 30 days. Biochemical studies as well as light microscopy (LM) and scanning electron microscopy (SEM) revealed significant metal exposure-induced alterations in gills. Besides ultastructural changes, activities of antioxidant enzymes such catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD) were significantly altered in the gills of exposed fish. The reduced glutathione (GSH) was significantly (p<0.001) decreased, while lipid peroxidation (LPO) was significantly (p<0.001) increased. The main alterations in general morphology of fish gills included spiking and fusion of secondary lamellae, formation of club-shaped filaments, and vacuolization and necrosis of filament epithelium in the interlamellar regions. SEM studies showed gradual increase of the density and apical surface area of the chloride cells and transformation of the surface structure of the pavement cells. The results of this study indicate adaptive as well a toxic responses in fish gills exposed to mixture of trace metals. Low concentrations of trace metal appear to compromise the antioxidant defense of gills. Lesions in the gill morphology caused by the effect of low concentrations of trace metals could lead to functional alterations and interference with fundamental processes such as maintenance of osmoregulation, gas exchange and xenobiotic metabolism in the exposed fish populations.

  5. The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®

    PubMed Central

    Ganguly, Bishu; Balasa, Balaji; Efros, Lyubov; Hinton, Paul R.; Hartman, Stephen; Thakur, Archana; Xiong, Joanna M.; Schmidt, Brian; Robinson, Randy R.; Sornasse, Thierry; Vexler, Vladimir; Sheridan, James P.

    2016-01-01

    ABSTRACT The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax. PMID:27367933

  6. Selective staining of CdS on ZnO biolabel for ultrasensitive sandwich-type amperometric immunoassay of human heart-type fatty-acid-binding protein and immunoglobulin G.

    PubMed

    Qin, Xiaoli; Xu, Aigui; Liu, Ling; Sui, Yuyun; Li, Yunlong; Tan, Yueming; Chen, Chao; Xie, Qingji

    2017-05-15

    We report on an ultrasensitive metal-labeled amperometric immunoassay of proteins, which is based on the selective staining of nanocrystalline cadmium sulfide (CdS) on ZnO nanocrystals and in-situ microliter-droplet anodic stripping voltammetry (ASV) detection on the immunoelectrode. Briefly, antibody 1 (Ab1), bovine serum albumin (BSA), antigen and ZnO-multiwalled carbon nanotubes (MWCNTs) labeled antibody 2 (Ab2-ZnO-MWCNTs) were successively anchored on a β-cyclodextrin-graphene sheets (CD-GS) nanocomposite modified glassy carbon electrode (GCE), forming a sandwich-type immunoelectrode (Ab2-ZnO-MWCNTs/antigen/BSA/Ab1/CD-GS/GCE). CdS was selectively grown on the catalytic ZnO surfaces through chemical reaction of Cd(NO3)2 and thioacetamide (ZnO-label/CdS-staining), due to the presence of an activated cadmium hydroxide complex on ZnO surfaces that can decompose thioacetamide. A beforehand cathodic "potential control" in air and then injection of 7μL of 0.1M aqueous HNO3 on the immunoelectrode allow dissolution of the stained CdS and simultaneous cathodic preconcentration of atomic Cd onto the electrode surface, thus the following in-situ ASV detection can be used for immunoassay with enhanced sensitivity. Under optimized conditions, human immunoglobulin G (IgG) and human heart-type fatty-acid-binding protein (FABP) are analyzed by this method with ultrahigh sensitivity, excellent selectivity and small reagent-consumption, and the limits of detection (LODs, S/N=3) are 0.4fgmL(-1) for IgG and 0.3fgmL(-1) for FABP (equivalent to 73 FABP molecules in the 6μL sample employed).

  7. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

    PubMed Central

    Roben, P; Moore, J P; Thali, M; Sodroski, J; Barbas, C F; Burton, D R

    1994-01-01

    Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently. Images PMID:7518527

  8. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial

    PubMed Central

    Ackerman, Margaret; Saunders, Kevin O.; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Michael, Nelson L.; O’Connell, Robert J.; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Phogat, Sanjay; Alam, S. Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S.; Montefiori, David C.; Harrison, Stephen C.; Haynes, Barton F.

    2017-01-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. Trial Registration: ClinicalTrials.gov NCT01435135 PMID:28235027

  9. Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

    PubMed Central

    Hershkoviz, R; Greenspoon, N; Mekori, Y A; Hadari, R; Alon, R; Kapustina, G; Lider, O

    1994-01-01

    The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition. PMID:7905794

  10. Key Role of Effector Memory CD4+ T Lymphocytes in a Short-Incubation Heparin-Binding Hemagglutinin Gamma Interferon Release Assay for the Detection of Latent Tuberculosis

    PubMed Central

    Wyndham-Thomas, Chloé; Corbière, Véronique; Dirix, Violette; Smits, Kaatje; Domont, Fanny; Libin, Myriam; Loyens, Marc; Locht, Camille

    2014-01-01

    The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4+ T lymphocytes, a finding suggestive of continuous HBHA presentation during latency. PMID:24391135

  11. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.

    PubMed

    Easterhoff, David; Moody, M Anthony; Fera, Daniela; Cheng, Hao; Ackerman, Margaret; Wiehe, Kevin; Saunders, Kevin O; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Kim, Jerome; Michael, Nelson L; O'Connell, Robert J; Excler, Jean-Louis; Robb, Merlin L; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Tartaglia, James; Phogat, Sanjay; Kepler, Thomas B; Alam, S Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S; Montefiori, David C; Tomaras, Georgia D; Harrison, Stephen C; Haynes, Barton F

    2017-02-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.

  12. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    PubMed

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  13. Parallel developmental genetic features underlie stickleback gill raker evolution

    PubMed Central

    2014-01-01

    Background Convergent evolution, the repeated evolution of similar phenotypes in independent lineages, provides natural replicates to study mechanisms of evolution. Cases of convergent evolution might have the same underlying developmental and genetic bases, implying that some evolutionary trajectories might be predictable. In a classic example of convergent evolution, most freshwater populations of threespine stickleback fish have independently evolved a reduction of gill raker number to adapt to novel diets. Gill rakers are a segmentally reiterated set of dermal bones important for fish feeding. A previous large quantitative trait locus (QTL) mapping study using a marine × freshwater F2 cross identified QTL on chromosomes 4 and 20 with large effects on evolved gill raker reduction. Results By examining skeletal morphology in adult and developing sticklebacks, we find heritable marine/freshwater differences in gill raker number and spacing that are specified early in development. Using the expression of the Ectodysplasin receptor (Edar) gene as a marker of raker primordia, we find that the differences are present before the budding of gill rakers occurs, suggesting an early change to a lateral inhibition process controlling raker primordia spacing. Through linkage mapping in F2 fish from crosses with three independently derived freshwater populations, we find in all three crosses QTL overlapping both previously identified QTL on chromosomes 4 and 20 that control raker number. These two QTL affect the early spacing of gill raker buds. Conclusions Collectively, these data demonstrate that parallel developmental genetic features underlie the convergent evolution of gill raker reduction in freshwater sticklebacks, suggesting that even highly polygenic adaptive traits can have a predictable developmental genetic basis. PMID:24851181

  14. Gill histopathologies following exposure to nanosilver or silver nitrate.

    PubMed

    Hawkins, Adam D; Thornton, Cammi; Kennedy, Alan J; Bu, Kaixuan; Cizdziel, James; Jones, Bradley W; Steevens, Jeffery A; Willett, Kristine L

    2015-01-01

    Fish gill is the site for many crucial physiological functions. It is among the first sites of xenobiotic exposure, and gill histopathological alterations may be detected soon after toxicant exposure. Silver (Ag) is one of the most toxic metals to aquatic organisms mainly due to its ability to disrupt ionic regulation. The goal of this study was to determine the effect of ionic and nanoscale Ag on fathead minnow gills by examining gill histology and Na(+)/K(+)-ATPase immunoreactivity. Fathead minnows were exposed to two measured concentrations of silver nitrate (AgNO3: 1.3 or 3.7 μg/L as Ag(+)), citrate silver nanoparticles (citrate-AgNP: 15 or 39 μg/L), and polyvinylpyrrolidone-AgNP (PVP-AgNP) (AgNP: 11 or 50 μg/L). Circulatory disturbances were the most prevalent gill alterations detected and were significantly increased in all Ag treatment groups compared to control. AgNO3 (1.3 μg/L) was the only treatment that significantly elevated the number of total mucous goblet cells present. In all other Ag treatments, the percent of degenerated goblet cells was significantly increased compared to control. When the sum of all histopathological abnormalities (weighted index) was calculated, all Ag groups displayed a significantly higher index, with citrate-AgNP having the highest toxicity (index of 10 ± 0.32 versus 2.4 ± 0.6 in controls). Gill Na(+)/K(+)-ATPase immunoreactivity was decreased by Ag. These results indicated that both AgNO3 and AgNP created similar disruptions in gill structure and ionic regulation, possibly due to the ionic Ag portion of each treatment.

  15. The nephridial hypothesis of the gill slit origin.

    PubMed

    Ezhova, Olga V; Malakhov, Vladimir V

    2015-12-01

    Metameric gill slits are mysterious structures, unique for Chordata and Hemichordata, and also, perhaps, for the extinct Cambrian Calcichordata. There is a discussed hypothesis of the gill slits origin from the metameric nephridia. According to the hypothesis, the hypothetical metameric deuterostome ancestor had in each segment a pair of coelomoducts and a pair of intestinal pockets. In the anterior segments, the coelomoducts have fused with the intestinal pockets. As a result, each nephridium opened both into the gut and into the environment. Then the dissepiments and funnels reduced in all segments except the collar one. Thus, in recent enteropneusts, only the first pair of gill slits keeps the ancestral arrangement communicating at the same time with the gut, with the environment, and with the coelom of the preceding (collar) segment. In the anterior part of the branchio-genital trunk region of enteropneusts, the metameric intestinal pockets remained, as well as the metameric coelomoducts functioning as the ducts of the metameric gonads, i.e., as the gonoducts. The consequence of the hypothesis is that the metameric gill pores originate from the metameric excreting pores, and the metameric branchial sacs originate from the metameric endodermal pockets of the gut fused with the coelomoducts. The metameric gill slits by themselves correspond with metameric openings connecting the gut with metameric intestinal pockets. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 647-652, 2015. © 2015 Wiley Periodicals, Inc.

  16. Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricus.

    PubMed

    Company, R; Serafim, A; Bebianno, M J; Cosson, R; Shillito, B; Fiala-Médioni, A

    2004-01-01

    Metals are known to influence lipid peroxidation and oxidative status of marine organisms. Hydrothermal vent mussels Bathymodiolus azoricus live in deep-sea environments with anomalous conditions, including high metal concentrations. Although B. azoricus are aerobic organisms they possess abundant methano and thioautotrophic symbiotic bacteria in the gills. The enzymatic defences (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (Total GPx) and selenium-dependent glutathione peroxidase (Se-GPx)) and lipid peroxidation were determined in the gills of B. azoricus exposed to Cd (0.9 microM), Cu (0.4 microM) and Hg (0.1 microM) with different times of exposure. The experiments were performed in pressurized containers at 9+/-1 degrees C and 85 bars. Results show that vent mussels possess antioxidant enzymatic protection in the gills. Cd and Cu had an inhibitory effect in the enzymatic defence system, contrarily to Hg. These enzymatic systems are not completely understood in the B. azoricus, since reactive oxygen species might be produced through other processes than natural redox cycling, due to hydrogen sulphide and oxygen content present. Also the symbiotic bacteria may play an important contribution in the antioxidant protection of the gills.

  17. Human diffusely adhering Escherichia coli expressing Afa/Dr adhesins that use human CD55 (decay-accelerating factor) as a receptor does not bind the rodent and pig analogues of CD55.

    PubMed

    Hudault, Sylvie; Spiller, O Brad; Morgan, B Paul; Servin, Alain L

    2004-08-01

    Afa/Dr diffusely adhering Escherichia coli (DAEC) bacteria that are responsible for recurrent urinary tract and gastrointestinal infections recognized as a receptor the glycosylphosphatidylinositol (GPI)-anchored protein decay-accelerating factor (DAF; CD55) at the brush border of cultured human intestinal cells. Results show that Afa/Dr DAEC C1845 bacteria were poorly associated with the mucosa of the gastrointestinal tract of infected mice. We conducted experiments with Chinese hamster ovary (CHO) cells stably transfected with mouse (GPI or transmembrane forms), pig, or human CD55 or mouse Crry cDNAs or transfected with empty vector pDR2EF1 alpha. Recombinant E. coli AAEC185 bacteria expressing Dr or F1845 adhesins bound strongly to CHO cells expressing human CD55 but not to the CHO cells expressing mouse (transmembrane and GPI anchored), rat, or pig CD55 or mouse Crry. Positive clustering of CD55 around Dr-positive bacteria was observed in human CD55-expressing CHO cells but not around the rarely adhering Dr-positive bacteria randomly distributed at the cell surface of CHO cells expressing mouse, rat, or pig CD55.

  18. Bioconcentration of zinc and its effect on the biochemical constituents of the gill tissues of Labeo rohita: An FT-IR study

    NASA Astrophysics Data System (ADS)

    Palaniappan, PL. RM.; Nishanth, T.; Renju, V. B.

    2010-03-01

    In the present work, an attempt has been made to assess the bioconcentration and distribution of zinc on the selected organs of Labeo rohita and to study the effect of zinc exposure on the biochemical constitutions of gill tissues of L. rohita by using FT-IR Spectroscopy. The concentration pattern in the organs reveals that the liver is the prime site of metal binding and muscle accumulates least metal concentration. The accumulation profile is in the order: liver > gill > kidney > brain > bone > muscle. It has also been observed that the administration of chelating agent D-Penicillamine (DPA) reduces the zinc concentration in all tissues more effectively than the administration of the chelating agent Ethylene Diamine Tetra Acetic acid. The FT-IR spectra reveal that zinc exposure causes significant changes in the biochemical constitutions of the gill tissues. It causes an alteration in the protein secondary structures by decreasing the α-helix and increasing the β-sheet contents. Further, it has been observed that the administration of chelating agent DPA improves the protein and lipid contents in the gill tissues compared to zinc exposed tissues. This result shows that DPA is the effective chelator of zinc in reducing the body burden of L. rohita fingerlings. In conclusion, the findings of the current study suggest that zinc exposure causes significant changes in both lipids and proteins of the gill tissues, and changes the protein profile in favour of β-sheet structure.

  19. Teleost chloride cell. II. Autoradiographic localization of gill Na,K- ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments

    PubMed Central

    1976-01-01

    The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport. PMID:132451

  20. High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6

    PubMed Central

    Xu, B; Broome, U; Ericzon, B-G; Sumitran-Holgersson, S

    2002-01-01

    Aim: Sera of patients with autoimmune liver diseases were investigated for the presence of autoantibodies binding to human biliary epithelial cells (BECs). Furthermore, their functional capacity was investigated by testing their capacity to fix complement as well as induce expression of various adhesion molecules and production of cytokines. Methods: Sera from patients with various stages of primary sclerosing cholangitis (PSC; n=30), primary biliary cirrhosis (PBC; n=29), autoimmune hepatitis (AIH; n=25), and normal controls (n=12) were investigated for the presence of antibodies that reacted with unstimulated and cytokine stimulated BECs isolated from a normal healthy liver. To demonstrate organ specificity, lung epithelial cells (LECs) were used as control cells. Antibodies were tested for their functional capacity. Results: Compared with controls (8%), significantly higher numbers of PSC patients (63%, p=0.001), but not PBC (37%, NS) or AIH (16%, NS) patients, had anti-BEC antibodies. In 90% of PSC patients, the autoantibodies reacted only with cytokine stimulated target cells. Lower numbers of PSC (6%), PBC (10%), and AIH (0%) patients had LEC antibodies. Other significant findings were that anti-BEC antibodies were found in (i) PSC patients with either the HLA-DRB1*0301 or DR2 allele compared with those without (p=0.007); and (ii) in PBC patients with end stage disease compared with those without (p=0.018). Furthermore, anti-BEC antibodies from PSC and PBC but not AIH patients induced BECs to produce high levels of the cytokine interleukin 6. IgM and IgG fractions isolated from PSC but not PBC and AIH sera induced significantly increased expression of the cell adhesion molecule CD44. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis of BEC membranes demonstrated a specific band of 40 kDa with PSC sera and 45, 42, 30, and 33 kDa bands with PBC sera, which were absent in control groups. Conclusion: Thus for the first time we

  1. Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC)

    PubMed Central

    Ohana, Ofra Malka; Ozer, Janet; Prinsloo, Isebrand; Benharroch, Daniel; Gopas, Jacob

    2015-01-01

    Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (LewisX), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system. PMID:26418972

  2. Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC).

    PubMed

    Ohana, Ofra Malka; Ozer, Janet; Prinsloo, Isebrand; Benharroch, Daniel; Gopas, Jacob

    2015-01-01

    Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (Lewis(X)), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

  3. IFN-gamma and prostaglandin E2 inhibit IL-4-induced expression of Fc epsilon R2/CD23 on B lymphocytes through different mechanisms without altering binding of IL-4 to its receptor

    SciTech Connect

    Galizzi, J.P.; Cabrillat, H.; Rousset, F.; Menetrier, C.; de Vries, J.E.; Banchereau, J.

    1988-09-15

    Human rIL-4 specifically induces the expression of the low affinity receptor for IgE (Fc epsilon R2/CD23) on normal B cells and on the Burkitt lymphoma cell line Jijoye. IL-4 does not induce the generation of the second messenger cAMP in Jijoye cells. PGE2 (at 10(-7) M) was found to inhibit by 50% the IL-4 mediated Fc epsilon R2/CD23 induction on Jijoye cells. The PGE2 half maximum inhibitory concentration (1 nM) was comparable to that inducing a half maximal increase of intracellular cAMP (4nM PGE2). 8-bromo-cAMP (10(-3) M), forskolin (10(-5) M), and cholera toxin (100 ng/ml), which increase intracellular cAMP levels, also inhibited by 40 to 80% the IL-4 induced Fc epsilon R2/CD23 expression on Jijoye cells. PGE2 8-bromo-cAMP, forskolin, and cholera toxin also inhibited the IL-4-induced Fc epsilon R2/CD23 expression on normal B lymphocytes. Taken together these data suggest that PGE2 inhibits the IL-4 induced Fc epsilon R2/CD23 through an increase of intracellular cAMP. In contrast, IFN-gamma, which strongly inhibits IL-4-mediated Fc epsilon R2/CD23 expression on Jijoye cells, did not increase intracellular cAMP levels and thus probably acts through another mechanism. IFN-gamma and PGE2 did not inhibit binding of IL-4 to its receptor. It could be excluded that IFN-gamma and PGE2 were acting via an alteration/desensitization of the IL-4R inasmuch as 24 h pre-incubation of Jijoye cells with these agents affected neither the affinity of 125I-IL-4 for its receptor (Kd = 0.8 to 1.5 x 10(-10) M) nor the maximal number of binding sites per Jijoye cells (Bmax = 390 to 550). Furthermore, IFN-gamma and PGE2 did not affect the internalization and degradation of 125I-IL-4. These data demonstrate that PGE2 and IFN-gamma inhibit the IL-4-mediated induction of Fc epsilon R2/CD23 on B lymphocytes via different mechanisms that do not alter the interaction of IL-4 with its receptor.

  4. 1,25D3 prevents CD8+Tc2 skewing and asthma development through VDR binding changes to the Cyp11a1 promoter

    PubMed Central

    Schedel, Michaela; Jia, Yi; Michel, Sven; Takeda, Katsuyuki; Domenico, Joanne; Joetham, Anthony; Ning, Fangkun; Strand, Matthew; Han, Junyan; Wang, Meiqin; Lucas, Joseph J.; Vogelberg, Christian; Kabesch, Michael; O'Connor, Brian P.; Gelfand, Erwin W.

    2016-01-01

    Effector CD8+ T cells convert from IFN-γ+ (Tc1) to IL-13+ (Tc2) cells in the presence of IL-4. Underlying regulatory mechanisms are not fully defined. Here, we show that addition of 1,25D3, the active form of vitamin D3, during CD8+ T-cell differentiation prevents IL-4-induced conversion to IL-13-producers. Transfer of 1,25D3-treated CD8+ T cells into sensitized and challenged CD8+-deficient recipients fails to restore development of lung allergic responses. 1,25D3 alters vitamin D receptor (VDR) recruitment to the Cyp11a1 promoter in vitro and in vivo in the presence of IL-4. As a result, protein levels and enzymatic activity of CYP11A1, a steroidogenic enzyme regulating CD8+ T-cell conversion, are decreased. An epistatic effect between CYP11A1 and VDR polymorphisms may contribute to the predisposition to childhood asthma. These data identify a role for 1,25D3 in the molecular programming of CD8+ T-cell conversion to an IL-13-secreting phenotype through regulation of steroidogenesis, potentially governing asthma susceptibility. PMID:26750596

  5. Reconciling catch differences from multiple fishery independent gill net surveys

    USGS Publications Warehouse

    Kraus, Richard T.; Vandergoot, Christopher; Kocovsky, Patrick M.; Rogers, Mark W.; Cook, H. Andrew; Brenden, Travis O.

    2017-01-01

    Fishery independent gill net surveys provide valuable demographic information for population assessment and resource management, but relative to net construction, the effects of ancillary species, and environmental variables on focal species catch rates are poorly understood. In response, we conducted comparative deployments with three unique, inter-agency, survey gill nets used to assess walleye Sander vitreus in Lake Erie. We used an information-theoretic approach with Akaike’s second-order information criterion (AICc) to evaluate linear mixed models of walleye catch as a function of net type (multifilament and two types of monofilament netting), mesh size (categorical), Secchi depth, temperature, water depth, catch of ancillary species, and interactions among selected variables. The model with the greatest weight of evidence showed that walleye catches were positively associated with potential prey and intra-guild predators and negatively associated with water depth and temperature. In addition, the multifilament net had higher average walleye catches than either of the two monofilament nets. Results from this study both help inform decisions about proposed gear changes to stock assessment surveys in Lake Erie, and advance our understanding of how multispecies associations explain variation in gill net catches. Of broader interest to fishery-independent gill net studies, effects of abiotic variables and ancillary species on focal specie’s catch rates were small in comparison with net characteristics of mesh size or twine type.

  6. Identifying the Child with Gilles de la Tourette Syndrome.

    ERIC Educational Resources Information Center

    Anderson, Donna J.

    1993-01-01

    This article presents a brief introduction to Gilles de la Tourette Syndrome (a neuropsychiatric disorder characterized by motor and vocal tics and obsessive-compulsive behaviors). It describes the nature of the disorder, treatment, and service provision (evaluation and assessment and the Individual Education Plan). (DB)

  7. Matter in Motion: The Educational Materialism of Gilles Deleuze

    ERIC Educational Resources Information Center

    Cole, David R.

    2012-01-01

    This paper critically examines the materialism that Gilles Deleuze espouses in his oeuvre to the benefit of educational theory. In "Difference and Repetition", he presented transcendental empiricism by underwriting Kant with realism (Deleuze, 1994). Later, in "Capitalism & Schizophrenia I & II" that were co-written with Felix Guattari (1984, 1988)…

  8. Gilles Lecture: Ocular Motility in a Time of Paradigm Shift

    PubMed Central

    Demer, Joseph L.

    2007-01-01

    Recent progress in understanding of the structure and function of extraocular muscles, and our ability to image them clinically, allows prediction of revolutionary progress in diagnosis and treatment of strabismus in the coming decades. This perspective memorializes a lecture given in honor of Dr. William Gilles, who has for decades been the paternal leader of strabismology in southern Australia. PMID:17181611

  9. Host-guest inclusion systems of daidzein with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD): Preparation, binding behaviors and water solubility

    NASA Astrophysics Data System (ADS)

    Deng, Yinghui; Pang, Yanhua; Guo, Yafei; Ren, Yufeng; Wang, Fen; Liao, Xiali; Yang, Bo

    2016-08-01

    Daidzein is an isoflavone of naturally abundance existing in plants and foods which has attracted much attention for its significant benefits on human health. However, its application was severely limited by its poor solubilities, instability and low bioavailability. To overcome these drawbacks, inclusion complexes of daidzein with two cyclodextrin (CD) derivatives, i.e., 2-hydropropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) were prepared and characterized both in solution and solid state by 1D and 2D NMR, XRD, SEM and elemental analyses. Fluorescence spectroscopy and the Job plot were used to demonstrate a mainly 1:1 inclusion mode between daidzein and CDs. Their thermal stabilities were evaluated with TG and DSC experiments. Moreover, water solubility of daidzein was significantly improved by inclusion complexation with CDs. These results might suggest valuable approaches to developments of new pharmaceutical formulations of daidzein.

  10. Binding Strength and Dynamics of Invariant Natural Killer Cell T Cell Receptor/CD1d-Glycosphingolipid Interaction on Living Cells by Single Molecule Force Spectroscopy*

    PubMed Central

    Bozna, Bianca L.; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-01-01

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells. PMID:21454514

  11. Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

    PubMed

    Bozna, Bianca L; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-05-06

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

  12. Fatty acids from the cyanobacterium Microcystis aeruginosa with potent inhibitory effects on fish gill Na+/K+-ATPase activity.

    PubMed

    Bury, N R; Codd, G A; Wendelaaar Bonga, S E; Flik, G

    1998-01-01

    Fatty acids from two strains of the cyanobacterium Microcystis aeruginosa, PCC 7820 (a strain that produces the hepatotoxin microcystin-LR, MC-LR) and CYA 43 (a strain that produces only small quantities of MC-LR), were extracted, partially characterised and tested for their inhibitory effect on the K+-dependent p-nitrophenol phosphatase (pNPPase) activity of tilapia (Oreochromis mossambicus) gill basolateral membrane. Thin-layer chromatography of the lipids from dichloromethane:methanol extracts of M. aeruginosa PCC 7820 and CYA 43, using diethylether:isopropanol:formic acid (100:4.5:2.5) as solvent, yielded five inhibitory products from M. aeruginosa 7820 and six from M. aeruginosa CYA 43. None of these products could be related to MC-LR. The inhibitory behaviour of the products mimics that of a slow, tight-binding inhibitor. The inhibitory activity is removed by incubation of extracts with fatty-acid-free bovine serum albumin (FAF-BSA). However, FAF-BSA only partially reversed the inhibition of K+-dependent pNPPase on fish gills pre-exposed to the extracted products. We conclude that M. aeruginosa strains PCC 7820 and CYA 43 produce fatty acids with potent inhibitory effects on K+-dependent pNPPase. The release of these products following lysis of cyanobacterial blooms may help to explain fish kills through a disturbance of gill functioning.

  13. Binding of copper(II) ions to the polyproline II helices of PEVK modules of the giant elastic protein titin as revealed by ESI-MS, CD, and NMR.

    PubMed

    Ma, Kan; Wang, Kuan

    2003-10-01

    Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of

  14. Prognostic Value and Daily Trend of Interleukin-6, Neutrophil CD64 Expression, C-Reactive Protein and Lipopolysaccharide-Binding Protein in Critically Ill Patients: Reliable Predictors of Outcome or Not?

    PubMed Central

    Djordjevic, Dragan; Pejovic, Janko; Surbatovic, Maja; Jevdjic, Jasna; Radakovic, Sonja; Veljovic, Milic; Peric, Aneta; Andjelic, Tamara; Popovic, Nada

    2015-01-01

    Summary Background Severe sepsis and/or trauma complicated by multiple organ dysfunction syndrome are the leading causes of death in critically ill patients. The aim of this prospective single-centre study was to assess the prognostic value and daily trend of interleukin-6 (IL-6), neutrophil CD64 expression, C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP) regarding outcome in critically ill patients with severe trauma and/or severe sepsis. Outcome measure was hospital mortality. Methods One hundred and two critically ill patients admitted to the intensive care unit of a tertiary university hospital were enrolled in this prospective study. Blood samples were collected on admission (day 1), days 2 and 3. Results CD64 index was 1.6-fold higher on day 1 and 1.78-fold higher on day 2 in non-survivors (p<0.05). The area under the curve (AUC) for the CD64 index on day 1 for outcome was 0.727. At a cut-off level of 2.80 sensitivity was 75% and specificity was 65%. Patients with CD64 index level on day 1 higher than 2.80 had 2.4-fold higher probability of dying. Odds ratio is 2.40; 95% CI 0.60–9.67. Conclusions CD64 index on day 1 is a fairly good predictor of outcome. AUCs for IL-6, CRP and LBP were < 0.55, suggesting these biomarkers failed to predict outcome. PMID:28356852

  15. E-Cadherin, CD44v6, and Insulin-Like Growth Factor-II mRNA-Binding Protein 3 Expressions in Different Stages of Hydatidiform Moles.

    PubMed

    Wang, Jiajun; Zhao, Min; Xiao, Jianping; Wu, Man; Song, Yaohua; Yin, Yongxiang

    2016-09-01

    E-cadherin, CD44v6, and IMP3 expression in partial, complete, and invasive hydatidiform moles (HMs) was evaluated. High E-cadherin expression with low CD44v6 expression was observed in partial, complete, and invasive HMs, as well as in normal placental tissues; and there was no significant difference in E-cadherin and CD44v6 expression among the four groups. However, IMP3 expression was gradually decreased in the order of normal placental tissues, partial HMs, complete HMs, and invasive HMs; wherein, invasive HMs had the lowest level. Low IMP3 expression may serve as a prognostic biomarker for HMs, and IMP3 may play a certain role in HMs progression.

  16. Effects of Pb plus Cd mixtures on toxicity, and internal electrolyte and osmotic balance in the rainbow trout (Oncorhynchus mykiss).

    PubMed

    Clemow, Yvonne H; Wilkie, Michael P

    2015-04-01

    The physiological and toxicological effects of Cd and Pb have been thoroughly studied, but relatively little work has been done to determine how mixtures of these metals affect fishes in soft (<100 μmol L(-1)Ca(2+)) slightly acidic (pH ∼6) waters typical of many lakes in the Canadian Shield and other regions. Recently, it has been suggested that acute exposure to Cd plus Pb mixtures (3h) had greater than additive effects on both Ca(2+) and Na(+) influx, which could potentially exacerbate disturbances to ion balance and result in greater toxicity in rainbow trout (Oncorhynchus mykiss). The goal of the present study was to test this hypothesis by assessing the physiological and toxicological effects of Cd plus Pb mixtures over longer time periods (3-5 days), but at relatively low, more environmentally relevant concentrations of these metals. Accordingly, toxicity and measurements of blood acid-base regulation (PaO2, pHa), hematology (Ht, Hb, MCHC, and Protein), ionic composition (body ions and plasma Ca(2+), Na(+), Cl(-), osmolality), unidirectional Na(+) fluxes and branchial Na(+)/K(+)-ATPase activity were measured in rainbow trout exposed to Cd plus Pb mixtures. Experiments on rainbow trout, implanted with dorsal aortic catheters for repetitive blood sampling, demonstrated that exposure to Pb alone (26 nmol PbL(-1)) was less toxic than Cd alone (6 nmol CdL(-1)), which was much less toxic to the fish than a Cd plus Pb mixture (7 nmol CdL(-1) plus 45 nmol PbL(-1)), which led to greater than additive 80% mortality by 5d. Both Cd and Pb inhibited Na(+) influx over 3d exposure to the metals, which was partially offset by decreases in the diffusive efflux (outflux) of Na(+) across the gill. Despite an absence of detectable effects of Pb alone on plasma ion balance, Cd plus Pb mixtures exacerbated Cd-induced reductions in plasma Ca(2+) concentration, and resulted in pronounced reductions in plasma Na(+), Cl(-), and osmolality. No effects on Na(+)/K(+)-ATPase activity

  17. Heavy metals distribution in muscle, liver, kidney and gill of European catfish (Silurus glanis) from Italian Rivers.

    PubMed

    Squadrone, S; Prearo, M; Brizio, P; Gavinelli, S; Pellegrino, M; Scanzio, T; Guarise, S; Benedetto, A; Abete, M C

    2013-01-01

    The accumulation of heavy metals in freshwaters has direct consequences to man and ecosystem. Thus, in this study, the concentrations of mercury, cadmium, lead, arsenic and chromium in organs of the predator European catfish (Silurus glanis) were investigated. Samples were collected annually in five sites covering the area of the Po River (North Italy) between 2007 and 2009. Metals were differently distributed in the various organs, the highest concentrations of Hg were found in muscle and liver, Cd in kidney, Pb in gill and liver, as in muscle, and of Cr in gill and liver. Our survey found Hg exceeding the Maximum Levels (MLs) of 0.5 ppm in 18% of samples, while Pb and Cd were lower than the MLs set by European regulations in muscle tissues (1881/2006/EC and 629/2008/EC). Hg concentrations were significantly related to sampling stations studied, according to the presence of many industrial activities in the catchment area of Bormida and Tanaro Rivers. The finding that Hg did not fit food fish legislation limits indicated that S. glanis flesh might not be utilised for human consumption. A close monitoring of metals pollution is strongly recommended especially in piscivorous fish, cause their bioaccumulation capacity.

  18. Interaction of glutathione reductase with heavy metal: the binding of Hg(II) or Cd(II) to the reduced enzyme affects both the redox dithiol pair and the flavin.

    PubMed

    Picaud, Thierry; Desbois, Alain

    2006-12-26

    To determine the inhibition mechanism of yeast glutathione reductase (GR) by heavy metal, we have compared the electronic absorption and resonance Raman (RR) spectra of the enzyme in its oxidized (Eox) and two-electron reduced (EH2) forms, in the absence and the presence of Hg(II) or Cd(II). The spectral data clearly show a redox dependence of the metal binding. The metal ions do not affect the absorption and RR spectra of Eox. On the contrary, the EH2 spectra, generated by addition of NADPH, are strongly modified by the presence of heavy metal. The absorption changes of EH2 are metal-dependent. On the one hand, the main flavin band observed at 450 nm for EH2 is red-shifted at 455 nm for the EH2-Hg(II) complex and at 451 nm for the EH2-Cd(II) complex. On the other hand, the characteristic charge-transfer (CT) band at 540 nm is quenched upon metal binding to EH2. In NADPH excess, a new CT band is observed at 610 nm for the EH2-Hg(II)-NADPH complex and at 590 nm for EH2-Cd(II)-NADPH. The RR spectra of the EH2-metal complexes are not sensitive to the NADPH concentration. With reference to the RR spectra of EH2 in which the frequencies of bands II and III were observed at 1582 and 1547 cm-1, respectively, those of the EH2-metal complexes are detected at 1577 and 1542 cm-1, indicating an increased flavin bending upon metal coordination to EH2. From the frequency shifts of band III, a concomitant weakening of the H-bonding state of the N5 atom is also deduced. Taking into account the different chemical properties of Hg(II) and Cd(II), the coordination number of the bound metal ion was deduced to be different in GR. A mechanism of the GR inhibition is proposed. It proceeds primarily by a specific binding of the metal to the redox thiol/thiolate pair and the catalytic histidine of EH2. The bound metal ion then acts on the bending of the isoalloxazine ring of FAD as well as on the hydrophobicity of its microenvironment.

  19. Allorecognition of HLA-C Mismatches by CD8+ T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

    PubMed Central

    Bettens, Florence; Buhler, Stéphane; Tiercy, Jean-Marie

    2016-01-01

    HLA-C locus mismatches (MMs) are the most frequent class I disparities in unrelated hematopoietic stem cell transplantation (HSCT) and have a detrimental impact on clinical outcome. Recently, a few retrospective clinical studies have reported some variability in the immunogenicity of HLA-C incompatibilities. To get better insight into presumably permissive HLA-C MMs, we have developed a one-way in vitro mixed lymphocyte reaction (MLR) assay allowing to quantify activated CD56−CD137+CD8+ lymphocytes in HLA-C incompatible combinations. T cell-mediated alloresponses were correlated with genetic markers such as HLA-C mRNA expression and the number of amino acid (aa) MMs in the α1/α2 domains (peptide-binding region). Because of the high rate of HLA-DPB1 incompatibilities in HLA-A-, B-, C-, DRB1-, and DQB1-matched unrelated HSCT patient/donor pairs, the impact of HLA-DPB1 mismatching, a potential bystander of CD4+ T cell activation, was also considered. Heterogeneous alloresponses were measured in 63 HLA-C-mismatched pairs with a positive assay in 52% of the combinations (2.3–18.6% activated CTLs), representing 24 different HLA-A~B~DRB1~DQB1 haplotypes. There was no correlation between measured alloresponses and mRNA expression of the mismatched HLA-C alleles. The HLA-C*03:03/03:04 MM did not induce any positive alloresponse in five MLRs. We also identified HLA-C*02:02 and HLA-C*06:02 as mismatched alleles with lower immunogenicity, and HLA-C*14:02 as a more immunogenic MM. A difference of at least 10 aa residues known to impact peptide/T cell receptor (TCR) binding and a bystander HLA-DPB1 incompatibility had a significant impact on CTL alloreactivity (p = 0.021). The same HLA-C MM, when recognized by two different responders with the same HLA haplotypes, was recognized differently, emphasizing the role of the T-cell repertoire of responding cells. In conclusion, mismatched HLA-C alleles differing by 10 or more aas in the peptide/TCR-binding region, when

  20. Aluminum bioconcentration at the gill surface of juvenile Atlantic salmon in acidic media

    SciTech Connect

    Wilkinson, K.J.; Campbell, P.G.C. . INRS-Eau)

    1993-11-01

    Aluminum uptake by Atlantic salmon was examined in the laboratory at pH 4.5, under conditions similar to those found in running waters on the Canadian Precambrian Shield during spring snowmelt. Gill uptake of Al was slow, approaching steady state only after 3 d of exposure. The greatest fraction of gill-associated Al was sorbed not to the gill surface itself, but to the gill mucus. Mucus appears to retard Al transport from solution to the membrane surface, thus delaying the acute biological response of the fish. Strongly associated gill [Al] was never greater than 10% of total gill Al in the early stages of the experiment indicated that this Al fraction could eventually exceed 50% of the total gill Al. In contrast to uptake, depuration of Al was extremely rapid; total gill [Al] of fish exposed to Al (pH 4.5) for 2 d decreased by 60% after only 2 h in an Al-free medium. The effect of fluoride complexation on Al bioconcentration was also examined. For equivalent Al[sup 3]+ concentrations, sorption of Al to the gill surface was higher in the presence of fluoride than in its absence, which suggests the formation of mixed ligand [F-Al-L-gill] complexes at the gill surface.

  1. Analysis of FcR non-binding anti-CD3 mAb in humanized mice identifies novel human gut tropic cells with regulatory function that are found in patients

    PubMed Central

    Waldron-Lynch, Frank; Henegariu, Octavian; Deng, Songyan; Preston-Hurlburt, Paula; Tooley, James; Flavell, Richard; Herold, Kevan C.

    2014-01-01

    The development and optimization of immune therapies in patients has been hampered by the lack of preclinical models in which their effects on human immune cells can be studied. As a result, observations that have been made in preclinical studies have suggested mechanisms of drug action in murine models that may not be confirmed in clinical studies. We have utilized a humanized mouse reconstituted with human hematopoetic stem cells to circumvent these limitations. We have studied the effects of teplizumab in this model, a Fc receptor non-binding humanized monoclonal anti-CD3 antibody that has been used to treat patients with Type 1 diabetes mellitus. A novel mechanism of action was identified where human gut tropic CCR6+ T cells leave the circulation and secondary lymph organs and migrate to the small intestine. They become producers of IL-10 which can be detected in the peripheral circulation. Blockade of migration of T cells to the small intestine by natalizumab abolishes the treatment effects of teplizumab. Direct translation of these findings was possible in patients with Type 1 diabetes treated with teplizumab since we found there is increased expression of IL-10 by CD4+CD25highCCR6+FoxP3 cells when they emerge into the peripheral circulation. These findings demonstrate that humanized mice may be used to identify novel immunologic mechanisms that occur in patients treated with immune modulators. PMID:22277969

  2. Modulation of HIV Binding to Epithelial Cells and HIV Transfer from Immature Dendritic Cells to CD4 T Lymphocytes by Human Lactoferrin and its Major Exposed LF-33 Peptide

    PubMed Central

    Carthagena, Laetitia; Becquart, Pierre; Hocini, Hakim; Kazatchkine, Michel D; Bouhlal, Hicham; Belec, Laurent

    2011-01-01

    Lactoferrin (LF), a multifunctional molecule present in human secretions, has potent inhibitory activities against human immunodeficiency virus (HIV). The aim of the study was to evaluate whether human LF (hLF) and its exposed domain LF-33 represented by the peptide (LF-33-GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP) involved in LF-HIV gag binding and endotoxines neutralization, may inhibit early steps of HIV mucosal transmission. Human LF and the peptide LF-33 inhibited the attachment of primary X4-tropic HIV-1NDK and R5-tropic HIV-1JR-CSF strains to human endometrial (HEC-1) and colorectal (HT-29) CD4-negative epithelial cells, the purified hLF being more potent (up to 80%) than the LF-33 peptide. In addition, the hLF, but not the LF-33 peptide, inhibited up to 40% the transfer in trans of HIV-1JR-CSF and HIV-1NDK, from immature dendritic cells to CD4 T lymphocytes, likely in a DC-SIGN-dependent manner. Altogether, these findings demonstrate that hLF can interfere with HIV-1 mucosal transmission by blocking virus attachment to epithelial cells and by inhibiting virus transfer from dendritic cells to CD4 T cells, two crucial steps of HIV dissemination from mucosae to lymphoid tissue. PMID:21660187

  3. Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: a review.

    PubMed

    Chasiotis, Helen; Kolosov, Dennis; Bui, Phuong; Kelly, Scott P

    2012-12-01

    Paracellular permeability characteristics of the fish gill epithelium are broadly accepted to play a key role in piscine salt and water balance. This is typically associated with differences between gill epithelia of teleost fishes residing in seawater versus those in freshwater. In the former, the gill is 'leaky' to facilitate Na(+) secretion and in the latter, the gill is 'tight' to limit passive ion loss. However, studies in freshwater fishes also suggest that varying epithelial 'tightness' can impact ionoregulatory homeostasis. Paracellular permeability of vertebrate epithelia is largely controlled by the tight junction (TJ) complex, and the fish gill is no exception. In turn, the TJ complex is composed of TJ proteins, the abundance and properties of which determine the magnitude of paracellular solute movement. This review provides consolidated information on TJs in fish gills and summarizes recent progress in research that seeks to understand the molecular composition of fish gill TJ complexes and what environmental and systemic factors influence those components.

  4. Famous people with Gilles de la Tourette syndrome?

    PubMed

    Monaco, Francesco; Servo, Serena; Cavanna, Andrea Eugenio

    2009-12-01

    Virtually no neurologist nor psychiatrist today can be unaware of the diagnosis of Gilles de la Tourette syndrome (GTS). Although the eponymous description by Dr. Georges Gilles de la Tourette was published in 1885, familiarity with this syndrome has been achieved only recently. In this article, the two most renown accounts of exceptional individuals retrospectively diagnosed with GTS are critically analyzed: British lexicographer Samuel Johnson and Austrian musician Wolfgang Amadeus Mozart. In both cases, clinical descriptions have been retrieved from written documents predating Gilles de la Tourette's original publication. The case for Samuel Johnson having GTS is strong, mainly based on Boswell's extensive biographical account. Johnson was reported to have a great range of tics and compulsions, including involuntary utterances, repetitive ejaculations, and echo-phenomena. On the other hand, there is circumstantial evidence that Mozart may have had hyperactivity, restlessness, sudden impulses, odd motor behaviors, echo/palilalia, love of nonsense words, and scatology, the latter being documented in autograph letters ("coprographia"). However, the evidence supporting the core features of GTS, i.e., motor and vocal tics, is rather inconsistent. Thus, GTS seems to be an implausible diagnosis in Mozart's medical history and completely unrelated to his undisputed musical genius.

  5. ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

    PubMed Central

    Nowyhed, Heba N.; Chandra, Shilpi; Kiosses, William; Marcovecchio, Paola; Andary, Farah; Zhao, Meng; Fitzgerald, Michael L.; Kronenberg, Mitchell; Hedrick, Catherine C.

    2017-01-01

    ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function. PMID:28091533

  6. Novel humanized anti-CD20 antibody BM-ca binds to a unique epitope and exerts stronger cellular activity than others.

    PubMed

    Kobayashi, Hideaki; Matsunaga, Yuka; Uchiyama, Yumiko; Nagura, Kenji; Komatsu, Yasuhiko

    2013-04-01

    Cellular activity of BM-ca, a novel humanized anti-CD20 antibody, was quantitatively compared with that of two other anti-CD20 antibodies used for clinical practice, rituximab and ofatumumab. The results of a complement-dependent cytotoxicity (CDC) assay revealed that the strongest antibody was ofatumumab, followed by BM-ca, with rituximab being the weakest. Ofatumumab and BM-ca were effective not only against rituximab-sensitive SU-DHL-4 cells but also against rituximab-resistant RC-K8 cells. In an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, although the effective concentrations against SU-DHL-4 cells were almost the same among these three antibodies, the maximum cytotoxic level was the highest for BM-ca. In an anti-cell proliferation assay using SU-DHL-4 cells, BM-ca was the most effective and ofatumumab, the weakest. Against RC-K8 cells, only BM-ca was effective. When combined with each of four cancer chemotherapeutics (prednisolone, vincristine, hydroxydaunorubicin, and cisplatin), BM-ca exerted the most effective combinatorial anti-cell proliferation activity. To assess the in vivo effect of BM-ca, we intravenously administered BM-ca into cynomolgus monkeys and found that the peripheral B-cell levels did not decrease in half of the animals. Sequencing of cDNA encoding CD20 of cynomolgus monkeys revealed that the responders and nonresponders had Leu/Pro (hetero) and Leu/Leu (homo) at amino acid (a.a.) position 160, respectively, suggesting that the epitope recognized by BM-ca was around this a.a. By analyzing reactivity to synthetic peptides, the epitope recognized by BM-ca was estimated to be a.a.'s 156-166, not shared with rituximab and ofatumumab. These results suggest BM-ca to be a promising anti-CD20 antibody having superior properties and recognizing a unique epitope.

  7. Effect of transforming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican.

    PubMed Central

    Romarís, M; Bassols, A; David, G

    1995-01-01

    We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7544118

  8. Resistances of antibiotics and heavy metals in Enterobacteriaceae spp. isolated from gills and intestines of Achanthobrama marmid (Heckel,1843) from Sir Dam lake Turkey.

    PubMed

    Toroglu, Sevil; Toroglu, Emin; Dincer, Sadik; Kara, Cemil; Kertmen, Metin

    2009-01-01

    A total of 94 bacteria, associated with wild Achanthobrama marmid (Heckel, 1843) in Sir Dam lake of Turkey identified. Subsequently selected isolates were characterized and identified to the genus level The 94 members of Enterobacteriaceae were isolated in the gills and intestines, and among the isolates, E. coli were represented at a rate of 55%, Shigella spp. at a rate of 21%, Salmonella spp. at a rate of 9%, Citrobacter spp. at a rate of 9%, Klebsiella spp. at a rate of 3% and Proteus spp., at a rate of 3%. A total of 94 bacteria resistant to antibiotics and heavy metals were isolated from total 47 of A. marmid samples and were investigated. Viable counts of antibiotic resistant bacteria isolated from gill and intestinal content samples showed high frequencies of resistance to Penicilline-G (KP) (68%), CZ (54%), FOX (48%), while the proportion of CRO (39%) and CTX (36%) resistance was low. In this research, heavy metal contamination in Sir Dam lake water samples and resistance frequency against heavy metals in isolated bacteria from gill and intestinal contents in A. marmid were investigated. Heavy metal contamination such as nickel (Ni), cadmium (Cd), copper (Cu) and chromium (Cr) determined diverse rate (except Mn) in water samples. The resistance frequency of the isolates was revealed different rate for the following heavy metals: Ni, Cd, Cu and Cr When the concentration of heavy metals increased, the resistance against heavy metals in diverse genus of isolates in different rate decreased.

  9. Esterase activity (EA), total oxidant status (TOS) and total antioxidant capacity (TAC) in gills of Mytilus galloprovincialis exposed to pollutants: Analytical validation and effects evaluation by single and mixed heavy metal exposure.

    PubMed

    Franco, Lorena; Romero, Diego; García-Navarro, José A; Teles, Mariana; Tvarijonaviciute, Asta

    2016-01-15

    The aims of the present study were to optimize and validate methods for esterase activity (EA), total oxidant status (TOS) and total antioxidant capacity (TAC) determination in mussel' gills, and to establish the relationships between these biomarkers and Pb, Cd and Cu pollution, in single form and ternary mixture. Two different buffers for sample homogenization, the need of ultracentrifugation, and analytical validation were evaluated. Coefficients of variation, when buffer without additives and ultracentrifugation were used, were <15%, and recovery were 97%-109% in all cases. The EA response tends to decrease with treatments, TOS decreased significantly in Cd and ternary groups, while TAC tended to increase in treatments with Pb, Cd and ternary groups. In conclusion, the methods for EA, TOS and TAC measurements in gills of mussel were precise and accurate and could be interesting resources in biomonitoring programmes.

  10. Pathological glutamatergic neurotransmission in Gilles de la Tourette syndrome.

    PubMed

    Kanaan, Ahmad Seif; Gerasch, Sarah; García-García, Isabel; Lampe, Leonie; Pampel, André; Anwander, Alfred; Near, Jamie; Möller, Harald E; Müller-Vahl, Kirsten

    2017-01-01

    Gilles de la Tourette syndrome is a hereditary, neuropsychiatric movement disorder with reported abnormalities in the neurotransmission of dopamine and γ-aminobutyric acid (GABA). Spatially focalized alterations in excitatory, inhibitory and modulatory neurochemical ratios within specific functional subdivisions of the basal ganglia, may lead to the expression of diverse motor and non-motor features as manifested in Gilles de la Tourette syndrome. Current treatment strategies are often unsatisfactory thus provoking the need for further elucidation of the underlying pathophysiology. In view of (i) the close spatio-temporal synergy exhibited between excitatory, inhibitory and modulatory neurotransmitter systems; (ii) the crucial role played by glutamate (Glu) in tonic/phasic dopaminergic signalling; and (iii) the interdependent metabolic relationship exhibited between Glu and GABA via glutamine (Gln); we postulated that glutamatergic signalling is related to the pathophysiology of Gilles de la Tourette syndrome. As such, we examined the neurochemical profile of three cortico-striato-thalamo-cortical regions in 37 well-characterized, drug-free adult patients and 36 age/gender-matched healthy control subjects via magnetic resonance spectroscopy at 3 T. To interrogate the influence of treatment on metabolite concentrations, spectral data were acquired from 15 patients undergoing a 4-week treatment with aripiprazole. Test-retest reliability measurements in 23 controls indicated high repeatability of voxel localization and metabolite quantitation. We report significant reductions in striatal concentrations of Gln, Glu + Gln (Glx) and the Gln:Glu ratio, and thalamic concentrations of Glx in Gilles de la Tourette syndrome in comparison to controls. ON-treatment patients exhibited no significant metabolite differences when compared to controls but significant increases in striatal Glu and Glx, and trends for increases in striatal Gln and thalamic Glx compared to baseline

  11. Binding of white spot syndrome virus to Artemia sp. cell membranes.

    PubMed

    Feng, Shuying; Li, Guangda; Feng, Wenpo; Huang, Jie

    2013-10-01

    Using differential velocity centrifugation, cell membranes of Artemia sp. were prepared, and their binding to white spot syndrome virus (WSSV) was analyzed in vitro. The results indicated that WSSV can specifically bind to Artemia cell membranes, and that WSSV receptor very likely existed in this membrane, which suggested that Artemia sp. may be a reservoir of WSSV. This study investigated the specific WSSV binding site by performing competitive inhibition experiments using shrimp gill cell membranes to bind WSSV to Artemia cell membranes. The results showed that shrimp gill cell membranes had a distinct inhibition effect on the specific binding of Artemia cell membranes to WSSV. Thus, potentially similar WSSV receptors or binding sites existed on Artemia sp. cell membranes and shrimp gill cell membranes. Taken together, these findings may provide experimental basis for the development of an effective approach to controlling WSSV, and theoretical basis for the study of WSSV receptors.

  12. Energy cost of NaCl transport in isolated gills of cutthroat trout.

    PubMed

    Morgan, J D; Iwama, G K

    1999-09-01

    Few studies have made direct estimates of the energy required for ion transport in gills of freshwater (FW) and seawater (SW) fish. Oxygen consumption was measured in excised gill tissue of FW-adapted cutthroat trout (Oncorhynchus clarki clarki) to estimate the energy cost of NaCl transport in that osmoregulatory organ. Ouabain (0.5 mM) and bafilomycin A1 (1 microM) were used to inhibit the Na+-K+ and H+ pumps, respectively. Both inhibitors significantly decreased gill tissue oxygen consumption, accounting for 37% of total tissue respiration. On a whole mass basis, the cost of NaCl uptake in the FW trout gill was estimated to be 1.8% of whole animal oxygen uptake. An isolated, saline-perfused gill arch preparation was also used to compare gill energetics in FW- and SW-adapted trout. The oxygen consumption of FW gills was significantly (33%) higher than SW gills. On a whole animal basis, total gill oxygen consumption in FW and SW trout accounted for 3.9 and 2.4% of resting metabolic rate, respectively. The results of both experiments suggest that the energy cost of NaCl transport in FW and SW trout gills represents a relatively small (<4%) portion of the animal's total energy budget.

  13. Morphology and ventilatory function of gills in the carpet shark family Parascylliidae (Elasmobranchii, Orectolobiformes).

    PubMed

    Goto, Tomoaki; Shiba, Yojiro; Shibagaki, Kazuhiro; Nakaya, Kazuhiro

    2013-06-01

    We examined gill morphology and ventilatory function in the carpet shark family Parascylliidae using 14 preserved specimens of Parascyllium ferrugineum, P. variolatum, P. collare and Cirrhoscyllium japonicum, and two live specimens of P. ferrugineum and P. variolatum. Morphological examinations revealed eight morphological characteristics related to the fifth gill, based on comparisons with other elasmobranchs, viz. large fifth gill slit without gill filaments, anatomical modifications in the fourth ceratobranchial cartilage and coraco-branchialis muscle, and the hypaxialis muscle associated with the fifth gill arch. Ventilation examinations using dyed seawater and prey items showed different water flows through the gill slits for respiration and prey-capture actions. For respiration, water sucked into the mouth was expelled equally through the first to fourth gill slits via a "double-pump" action, there being no involvement of the fifth gill slit. In prey-capture, however, water sucked into the mouth was discharged only via the widely opened fifth gill slit. This form of water flow is similar to that in other benthic suction-feeding sharks (e.g., Chiloscyllium plagiosum), except for the active water discharge by wide expansion and contraction of the fifth parabranchial cavity. The latter is dependent upon the morphological modifications of the fourth and fifth gill arches, derived phylogenetically as a mechanistic suction specialization in Parascylliidae.

  14. Gill Development and its functional and evolutionary implications in the blue mussel Mytilus edulis (Bivalvia: Mytilidae).

    PubMed

    Cannuel, Rozenn; Beninger, Peter G; McCombie, Helen; Boudry, Pierre

    2009-10-01

    Study of gill development in bivalve larvae and postlarvae provides information on the evolution of this organ and feeding mechanisms of early stages. Scanning electron microscopy was used to document the development of the filibranch homorhabdic gill in hatchery-reared larval, postlarval, and juvenile Mytilus edulis. Four key stages were identified during gill development: (1) transfer of the particle collection function from velum to gill at metamorphosis, with subsequent elongation of the gill filaments to form a gill basket, with complete frontal ciliation; (2) reflection of the inner demibranchs, and transition to a V-shaped gill; (3) delayed development of the outer demibranchs, occuring simultaneously along the gill axis, with transition to the adult final W-shape; and (4) formation of the ventral particle grooves and concomitant acquisition of dense abfrontal ciliation. These key stages signal shifts in the mechanisms of particle processing during the early development of M. edulis. Gill development in the homorhabdic filibranch M. edulis was similar to that of the early homorhabdic stages of the heterorhabdic filibranchs studied to date (Pectinidae), but different from that of the pseudolamellibranchs (Ostreidae), suggesting divergent evolution of this character. Similarly, the systems responsible for gill cohesion and structural integrity are common to both the homorhabdic and heterorhabdic filibranchs, suggesting evolutionary proximity, but they are patently different from those of the eulamellibranchs and pseudolamellibranchs, suggesting evolutionary divergence.

  15. The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling.

    PubMed

    Buonocore, Francesco; Randelli, Elisa; Bird, Steve; Secombes, Chris J; Costantini, Susan; Facchiano, Angelo; Mazzini, Massimo; Scapigliati, Giuseppe

    2006-04-01

    In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.

  16. PILRα binds an unknown receptor expressed primarily on CD56bright and decidual-NK cells and activates NK cell functions.

    PubMed

    Ophir, Yael; Duev-Cohen, Alexandra; Yamin, Rachel; Tsukerman, Pini; Bauman, Yoav; Gamliel, Moriya; Mandelboim, Ofer

    2016-07-05

    Natural Killer (NK) cells are innate immune lymphocytes specializing in recognition and killing of tumors and pathogens, using an array of activating and inhibitory receptors. NK inhibition is mediated by a large repertoire of inhibitory receptors, whereas a limited number of activating NK cell receptors execute NK cell activation. The ligands recognized by the activating receptors are stress-induced, pathogen derived, tumor specific and even self ligands. However, the full spectrum of NK cell receptors and ligands that control NK cell activity remains uncharacterized. Here we demonstrate that Paired Ig-Like type 2 Receptor Alpha (PILRα), binds a distinct human NK cell sub-population present in the peripheral blood and also in the decidua. We further demonstrate that the interaction of NK cells with PILRα expressing targets lead to elevated IFNγ secretion and cytotoxicity. In conclusion, we present here a novel NK activating ligand which binds and activates an unknown NK receptor expressed on a unique NK cell subset.

  17. Gill Na{sup +}, K{sup +}-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    SciTech Connect

    Brundage, S.; Jagoe, C.H.; Shaw-Allen, P.

    1995-12-31

    Active transport of Na{sup +} and K{sup +} for osmoregulation in fish involves gill Na{sup +}, K{sup +}-ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na{sup +}, K{sup +}-ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na{sup +}, K{sup +}-ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 {micro}g Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 {micro}g Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P <0.05 by Kruskal-Wallis test) less Hg (muscle and liver range 0.61--2.39 and 0.28--2.32 {micro}g Hg/g dry mass, respectively). In all reservoirs, liver Hg varied more among individuals than muscle Hg. Water chemistry was similar in all reservoirs. Fish from the three reservoirs did not differ significantly in gill ATPase activity, and a correlation between tissue Hg and Na{sup +}, K{sup +}-ATPase activity was not evident.

  18. Rapid activation of gill Na+,K+-ATPase in the euryhaline teleost Fundulus heteroclitus

    USGS Publications Warehouse

    Mancera, J.M.; McCormick, S.D.

    2000-01-01

    The rapid activation of gill Na+,K+-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na+,K+-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na+,K+-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na+,K+-ATPase, and the rapid increase after transfer to SW was not observed. Na+,K+-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na+,K+-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na+,K+-ATPase. Cycloheximide inhibited the increase in gill Na+,K+-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10-4 M > 10-5 M > 10-6 M). Actinomycin D or bumetanide in the culture (doses of 10-4 M, 10-5 M, and 10-6 M) did not affect gill Na+,K+-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na+,K+-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na+,K+-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. (C) 2000 Wiley-Liss, Inc.

  19. Six cases of Gilles de la Tourette's syndrome.

    PubMed

    Fernando, S J

    1976-05-01

    Six cases of Gilles de la Tourette's syndrome are discussed in the light of conflicting views on the aetiology of the condition. It is hypothesized that the onset of coprolalia in patients with persistent childhood tics indicates a disturbances of the normal balance between a need for tension relief by swearing and a capacity to control such vocal activity. Some conclusions are drawn on the management of the syndrome by the use of butyrophenones, massed practice of tics, and the promotion of personality development. A flexible approach geared to the individual patient's particular needs is recommended.

  20. Gill disease in marine farmed Atlantic salmon at four farms in Ireland.

    PubMed

    Rodger, H D; Murphy, K; Mitchell, S O; Henry, L

    2011-06-25

    A study of four marine salmon farms was undertaken in Ireland in 2008, with a focus on gill health and disease. All four farms suffered severe gill disease resulting in mortalities and, in some cases, failure to thrive. The aetiology of the gill pathologies in some cases was associated with small gelatinous zooplankton and bacteria, but also involved epitheliocystis and parasites such as marine costia (Ichthyobodo species) and amoebae (Neoparamoeba species). Treatments with oral broad-spectrum antibiotics and/or freshwater baths had equivocal benefits. There was a strong association of susceptibility to gill disease with one genetic strain of salmon.

  1. Investigation of the Cu binding site at [dCdG] and [CG] base pairs in the absence of a DNA backbone

    NASA Astrophysics Data System (ADS)

    Kim, Mi-Ji; Kim, Bo-Ra; Kim, Ho-Tae

    2011-03-01

    We investigated the gas-phase nucleobases [CGH] +1, [Cu(CG)] +1, [Cu(CG-H)] +1, and nucleoside [Cu(dCdG)] +1 complexes using collision-induced dissociation (CID) MS/MS spectra. Two forms, the Watson-Crick complex form (cation-base-base) and an interstrand complex form (base-cation-base), were included in the analysis of the [cation-base pair] complex structures. The main cytosine loss dissociation channel of the [Cu(CG)] +1 complex was analyzed as the loss of an interstrand [Cu(CG)] +1 form instead of the Watson-Crick form. The -43 amu (CONH) and -68 amu (cytosine-CONH) loss fragments of the [Cu(CG-H)] +1 complex can be explained by the dissociation process of the interstrand [Cu(CG-H)] +1 form.

  2. Effects of elevated seawater pCO2 on gene expression patterns in the gills of the green crab, Carcinus maenas

    PubMed Central

    2011-01-01

    Background The green crab Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO2 as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO2, Baltic Sea green crabs were exposed to a pCO2 of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR. Results Crabs responded mainly through fine scale adjustment of gene expression to elevated pCO2. However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO2 stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO2. However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl-/HCO3- exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study. Conclusions The moderate, but specific response of C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill

  3. Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils.

    PubMed

    Zen, Ke; Utech, Markus; Liu, Yuan; Soto, Illena; Nusrat, Asma; Parkos, Charles A

    2004-10-22

    The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.

  4. Structure and function of ionocytes in the freshwater fish gill.

    PubMed

    Dymowska, Agnieszka K; Hwang, Pung-Pung; Goss, Greg G

    2012-12-01

    Freshwater fishes lose ions to the external medium owing to the steep concentration gradients between the body fluids and the water. To maintain homeostasis, they use ionocytes to actively extract Na(+), Cl(-), and Ca(2+) from the dilute external medium and excrete acidic (H(+)) or basic (HCO(3)(-)) equivalents by specialized cells termed ionocytes that are responsible for transport of ions. Freshwater fishes have evolved diverse approaches to solving these similar ionic and acid-base problems. In the few well-studied species, there are clearly different patterns in the physiology and morphology for ionocytes in the gill. In this review, we describe the varying nomenclature of ionocytes that have been used in the past 80 years to allow direct comparison of ionocytes and their common functions in different species. We focus on the recent advancement in our understanding of the molecular mechanisms of ion and acid-base regulation as represented by ionocyte subtypes found in rainbow trout, killifish, tilapia and zebrafish gill.

  5. Subcellular localization and kinetic characterization of a gill (Na+, K+)-ATPase from the giant freshwater prawn Macrobrachium rosenbergii.

    PubMed

    França, Juliana L; Pinto, Marcelo R; Lucena, Malson N; Garçon, Daniela P; Valenti, Wagner C; McNamara, John C; Leone, Francisco A

    2013-07-01

    The stimulation by Mg(2+), Na(+), K(+), NH4 (+), and ATP of (Na(+), K(+))-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na(+), K(+))-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg(2+), Na(+), and K(+) concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V(M) = 115.0 ± 2.3 U mg(-1), K(0.5) = 0.10 ± 0.01 mmol L(-1). Stimulation by Na(+) (V(M) = 110.0 ± 3.3 U mg(-1), K(0.5) = 1.30 ± 0.03 mmol L(-1)), Mg(2+) (V(M) = 115.0 ± 4.6 U mg(-1), K(0.5) = 0.96 ± 0.03 mmol L(-1)), NH4 (+) (V(M) = 141.0 ± 5.6 U mg(-1), K(0.5) = 1.90 ± 0.04 mmol L(-1)), and K(+) (V(M) = 120.0 ± 2.4 U mg(-1), K(M) = 2.74 ± 0.08 mmol L(-1)) followed single saturation curves and, except for K(+), exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73% with K(I) = 12.4 ± 1.3 mol L(-1). Complementary inhibition studies suggest the presence of F0F1-, Na(+)-, or K(+)-ATPases, but not V(H(+))- or Ca(2+)-ATPases, in the gill microsomal preparation. K(+) and NH4(+) synergistically stimulated enzyme activity (≈25%), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4(+), and K(+) of the gill enzyme.

  6. Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

    PubMed

    Sarmiento, Katreena P; Panes, Vivian A; Santos, Mudjekeewis D

    2016-08-01

    Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp.

  7. Intracellular pH regulation in isolated trout gill mitochondrion-rich (MR) cell subtypes: evidence for Na+/H+ activity.

    PubMed

    Parks, Scott K; Tresguerres, Martin; Galvez, Fernando; Goss, Greg G

    2010-02-01

    We have studied intracellular pH (pH(i)) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH(4)Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na(+)-independent pH(i) recovery while the other cell type lacked a Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery were identified as PNA(+) (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA(-) (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na(+) provides a direct functional marker for PNA(+) and PNA(-) MR cells. Re-addition of Na(+) to acidified cells resulted in a transient pH(i) recovery in both cell types. This event was abolished by amiloride (500 microM) but it was insensitive to phenamil (50 microM). The phorbol ester PMA (1 microM) potentiated the Na(+) induced pH(i) recovery suggesting that activation by PKC is required for continuous Na(+)/H(+) exchanger activity in trout gill MR cells. This study is the first functional description of pH(i) recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pH(i) regulation in the gill epithelium.

  8. Identification and characterization of CD133+CD44+ cancer stem cells from human laryngeal squamous cell carcinoma cell lines

    PubMed Central

    Wang, Jue; Wu, Yongyan; Gao, Wei; Li, Fei; Bo, Yunfeng; Zhu, Meixia; Fu, Rong; Liu, Qingqing; Wen, Shuxin; Wang, Binquan

    2017-01-01

    Background: Laryngeal squamous cell carcinoma ranks second among head and neck squamous-cell carcinomas. Cancer stem cells can support cancer growth and malignant behavior. Therefore, cancer stem cells isolated from laryngeal squamous cell carcinoma tissue could be used to investigate the initiation, progression, and treatment strategies of this cancer. Methods: We isolated CD133-CD44-, CD133-CD44+, CD133+CD44- and CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep2 and TU-177 by magnetic-activated cell sorting. Sphere formation, cell proliferation, migration, invasion, colony formation, resistance to radio- and chemotherapy, and in vivo tumorigenicity of these populations were evaluated. Moreover, we investigated the expression of the stem-cell markers (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) in CD133-CD44-, CD133-CD44+, CD133+CD44-, CD133+CD44+ cell populations and parental Hep2 and TU-177 cells. Results: As compared with CD133-CD44-, CD133-CD44+, CD133+CD44- populations and parental cells, CD133+CD44+ cells showed higher cell viability, migration and invasive capability and colony formation ability as well as stronger resistance to cisplatin and irradiation. Moreover, levels of SOX2 and OCT4 and tumorigenicity in nude mice were greater in CD133+CD44+ Hep2 and TU-177 cells than other cell populations and parental cells. Conclusion: The CD133+CD44+ population of laryngeal squamous-cell carcinoma Hep2 and TU-177 cells have stem cell properties and showed more malignant features than CD133+CD44- and CD133-CD44+ cell populations. CD133+CD44+ cancer stem cells may be a promising target for developing anticancer drugs and treatment strategies for laryngeal squamous cell carcinoma. PMID:28261352

  9. Transciptome Analysis of the Gill and Swimbladder of Takifugu rubripes by RNA-Seq

    PubMed Central

    Cui, Jun; Liu, Shikai; Zhang, Bing; Wang, Hongdi; Sun, Hongjuan; Song, Shuhui; Qiu, Xuemei; Liu, Yang; Wang, Xiuli; Jiang, Zhiqiang; Liu, Zhanjiang

    2014-01-01

    The fish gill, as one of the mucosal barriers, plays an important role in mucosal immune response. The fish swimbladder functions for regulating buoyancy. The fish swimbladder has long been postulated as a homologous organ of the tetrapod lung, but the molecular evidence is scarce. In order to provide new information that is complementary to gill immune genes, initiate new research directions concerning the genetic basis of the gill immune response and understand the molecular function of swimbladder as well as its relationship with lungs, transcriptome analysis of the fugu Takifugu rubripes gill and swimbladder was carried out by RNA-Seq. Approximately 55,061,524 and 44,736,850 raw sequence reads from gill and swimbladder were generated, respectively. Gene ontology (GO) and KEGG pathway analysis revealed diverse biological functions and processes. Transcriptome comparison between gill and swimbladder resulted in 3,790 differentially expressed genes, of which 1,520 were up-regulated in the swimbladder while 2,270 were down-regulated. In addition, 406 up regulated isoforms and 296 down regulated isoforms were observed in swimbladder in comparison to gill. By the gene enrichment analysis, the three immune-related pathways and 32 immune-related genes in gill were identified. In swimbladder, five pathways including 43 swimbladder-enriched genes were identified. This work should set the foundation for studying immune-related genes for the mucosal immunity and provide genomic resources to study the relatedness of the fish swimbladder and mammalian lung. PMID:24454879

  10. Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus.

    PubMed

    Hyndman, Kelly A; Evans, David H

    2009-01-01

    We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.

  11. ANALYTICAL METHOD FOR THE DETERMINATION OF PHENYLGLUCURONIDE IN RAINBOW TROUT GILL WATER

    EPA Science Inventory

    Phenylglucuronide (PG), a primary phase II metabolite of phenol, can be excreted by fish through urine and feces, similar to mammals. In addition, it may also be possible to eliminate it through a fish's gills. In order to assess the significance of gill water elimination, analyt...

  12. Element ratios between digestive gland and gill tissues of the Antarctic bivalve Laternula elliptica as a proxy for element uptake from different environmental sources

    NASA Astrophysics Data System (ADS)

    Poigner, H.; Monien, D.; Monien, P.; Kriews, M.; Brumsack, H.-J.; Wilhelms-Dick, D.; Abele, D.

    2012-04-01

    Trace metals in bivalve carbonate shells are frequently used as environmental or paleoclimate proxies. Carbonate mineralogy and animals' physiology affect the incorporation of elements from different environmental sources into bivalve shells. Generally, metals from particulate matter are assimilated via the digestive tract; whereas dissolved metals are absorbed via gills. Therefore, measurements of element concentrations deposited in the shell matrix do not necessarily allow inference with respect to the assimilation pathways. In this study, we used element ratios between digestive gland (DG) and gills (cDG/cGill) of the Circum-Antarctic clam Laternula elliptica to identify predominating assimilation pathways and potential sources of bio-available metals. This normalization between tissues of each individual eliminates the effects of individual age and physiological condition (e.g. accumulation over lifetime, metabolic activity) on metal assimilation. These effects also minimize the reproducibility, when absolute element concentrations are compared between individuals from different locations. Therefore, an additional normalization is required. We favored "ellipsoid shell volume" over shell length or soft tissue weight as more conservative approximation for intra- and intersite comparisons. Metal concentrations in DG, gills, and hemolymph of the bivalve L. elliptica, collected at Potter Cove (King George Island, Antarctic Peninsula), were analyzed by means of inductively coupled plasma - optical emission spectroscopy and mass spectrometry after total acid digestion. The element ratios (cDG/cGill) indicate a predominant assimilation of Al, Ca, Fe, K, Mn, and Mg from the dissolved phase. These high Al and Fe concentrations in gill tissues and hemolymph are in contrast to the low solubility of Al and Fe in seawater. But high dissolved Fe concentrations in pore waters (up to 1400 μg L-1 due to suboxic sediment conditions) and glacial melt waters enriched in dissolved

  13. The yellow catfish, Pelteobagrus fulvidraco (Siluriformes) metallothionein cDNA: molecular cloning and transcript expression level in response to exposure to the heavy metals Cd, Cu, and Zn.

    PubMed

    Kim, Jin-Hyoung; Rhee, Jae-Sung; Dahms, Hans-Uwe; Lee, Young-Mi; Han, Kyung-Nam; Lee, Jae-Seong

    2012-10-01

    Metallothionein (MT) has been used extensively as a potential molecular biomarker to detect heavy metal pollution in aquatic organisms. In order to investigate the modulation effect of heavy metals and to establish suitable biomarkers for the monitoring of heavy metal pollution, Pelteobagrus fulvidraco metallothionein gene was characterized as the first report in the family Bagridae. Pf-MT transcript was detected at high levels in liver, gonad, kidney, and brain compared to other tissues. A time-course study in response to waterborne Cd (5 ppm) revealed that a significant increase in the Pf-MT transcript abundance was observed at 6 h in gill, kidney, and liver. These elevated levels were kept for 96 h, implying that Cd distributed fast into different organs and was involved in the tissue-specific induction pattern. We observed a significant Pf-MT transcript increase in liver tissues at 48 h, followed by gill at 12 h and intestine at 48 h after Cd exposure. This indicates hepatic MT expression as a potential biomarker of acute Cd exposure in this species. Cd-binding ability of recombinant Pf-MT protein provided evidence for sensitivity to Cd and other heavy metal exposure. In the case of Zn exposure (1 ppm), a significant increase in Pf-MT transcript abundance was observed at 12 h, and a peak induction level reaching sixfold at 24 h was kept until 48 h, showing similar transcript induction patterns with Cd. A high level of Pf-MT mRNA after exposure to Cu (1 ppm) was observed at 12 h that gradually increased until 96 h with a 12-fold induction, revealing a long-lasting induction and somewhat dissimilar pattern compared to other metals in liver. Our results demonstrate that Pf-MT can be induced by heavy metals in a tissue-specific and metal-specific manner and plays probably a conserved role in metal detoxification. This study provides new information on P. fulvidraco metallothionein gene for the use of biomarkers indicating metal pollution in fish.

  14. A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

    PubMed

    Gillis, J Andrew; Hall, Brian K

    2016-04-15

    Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis.

  15. Effects of sublethal copper concentrations on gills of white shrimp (Litopenaeus vannamei, Boone 1931).

    PubMed

    Soegianto, Agoes; Irawan, Bambang; Usman, Nuhman

    2013-12-01

    The objective of this study was to measure the copper (Cu) concentration in gills of juveniles Litopenaeus vannamei after exposure to Cu at sublethal concentrations, and to evaluate its effect upon the structure of gill tissue. The Cu concentration in gills of control shrimp was 0.075 mg/kg. Copper concentrations increased significantly by 147 %, 180 % and 205 % in gills of shrimp exposed to 0.675, 1.325 and 2.010 mg Cu/L, respectively. After exposure to 0.675 mg Cu/L for 15 days, gill tissue hyperplasia was observed, with a narrowing of the hemolymphatic lacunae. Necrosis and loss of hemolymphatic lacunae were observed at exposures of 1.325 and 2.010 mg Cu/L.

  16. Glycoproteins histochemistry of the gills of Odontesthes bonariensis (Teleostei, Atherinopsidae).

    PubMed

    Díaz, A O; García, A M; Escalante, A H; Goldemberg, A L

    2010-11-01

    The histochemistry of glycoproteins (GP) in the mucous cells of the gills of the silverside Odontesthes bonariensis was identified with: (1) oxidizable vicinal diols; (2) sialic acid and some of their chain variants, carbon 7 ((7) C), carbon 8 ((8) C) or carbon 9 ((9) C); (3) sialic acid residues without O-acyl substitution and with O-acyl substitution at (7) C, (8) C or (9) C; (4) carboxyl groups and (5) sulphate groups. A battery of seven biotinylated lectins allowed GPs sugar residues to be distinguished. Mucous cells showed the presence of neutral, sulphated and sialylated GPs. Dolichos biflorus agglutinin (DBA) and Glycine max agglutinin (SBA) showed strong positive staining; Arachis hypogaea agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I) and Triticum vulgaris agglutinin (WGA) showed moderate staining, while Ulex europaeus agglutinin-I (UEA-I) was completely negative.

  17. The systematic relevance of conidiogenesis modes in the gilled Agaricales.

    PubMed

    Walther, Grit; Garnica, Sigisfredo; Weiss, Michael

    2005-05-01

    Dikaryotic and haploid mycelia of more than 150 gilled species of euagarics were studied morphologically and by molecular phylogenetic methods. The morphological investigations revealed anamorphs in more than 90 species that were often specific at the genus or family level. Thallic conidiogenesis dominated and varied from fragmentation of normally branched hyphae to the formation of differentiated sympodially branched conidiophores. Secession modes, coiling of the conidiogenous hyphae or the swelling of the conidia were additional distinguishing features. Phylogenetic analysis of the D1-D3 domains of the nuclear gene for the ribosomal large subunit using a Bayesian Markov chain Monte Carlo approach resulted in several well-supported groups that are consistent with anamorph morphology. These results indicate that the anamorphs provide valuable characters for a natural classification of the Agaricales.

  18. Can the evolution of Cd resistance in prey alter Cd bioavailability to a predator?

    SciTech Connect

    Wallace, W.G.; Lopez, G.R.

    1995-12-31

    The deposit feeding oligochaete, Limnodrilus hoffmeisteri, inhabiting a Cd polluted cove on the Hudson River (Foundry Cove) has evolved resistance to Cd. The worms` resistance is attributed to the binding of Cd to metallothionein-like proteins (MT) and metal-rich granules (MRG). Their research focuses on determining whether Cd detoxification by oligochaetes alters Cd bioavailability and trophic transfer to a representative predator, the omnivorous grass shrimp Palaemonetes. Cd resistant and nonresistant worms were radiolabeled with the radioisotope Cd-109. After exposure to the metal, worm subcellular Cd-109 distributions were determined through differential centrifugation and biochemical fractionation. Absorption of Cd-109 by shrimp fed the radiolabeled worms was also determined. The authors show that Cd resistant worms have 30% of their Cd stored in MRG and 11% in the cytosol; nonresistant worms have 2% and 56% stored respectively. Both populations have about 65% of the Cd in the cytosol bound to a heat-stable (MT) fraction. The authors demonstrate that differences in worm subcellular Cd distributions translate into large differences in Cd trophic transfer. Shrimp fed Cd resistant worms absorb 22% of the ingested Cd; shrimp fed nonresistant worms absorb 76%. Their research demonstrates that through the efficient storage of Cd into insoluble-unbioavailable MRG, evolution of Cd resistance has effectively suppressed the bioavailability and trophic transfer of Cd.

  19. Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis).

    PubMed

    Hernroth, B; Baden, S; Tassidis, H; Hörnaeus, K; Guillemant, J; Bergström Lind, S; Bergquist, J

    2016-08-01

    Here, we aimed to investigate potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, as gills are directly facing seawater and the changing pH (predicted to be reduced from ∼8.1 to ∼7.7 by 2100). The AMP activity of gill and haemocyte extracts was compared at pH 6.0, 7.7 and 8.1, with a radial diffusion assay against Escherichia coli. The activity of the gill extracts was not affected by pH, while it was significantly reduced with increasing pH in the haemocyte extracts. Gill extracts were also tested against different species of Vibrio (V. parahaemolyticus, V. tubiashii, V. splendidus, V. alginolyticus) at pH 7.7 and 8.1. The metabolic activity of the bacteria decreased by ∼65-90%, depending on species of bacteria, but was, as in the radial diffusion assay, not affected by pH. The results indicated that AMPs from gills are efficient in a broad pH-range. However, when mussels were pre-exposed for pH 7.7 for four month the gill extracts presented significantly lower inhibit of bacterial growth. A full in-depth proteome investigation of gill extracts, using LC-Orbitrap MS/MS technique, showed that among previously described AMPs from haemocytes of Mytilus, myticin A was found up-regulated in response to lipopolysaccharide, 3 h post injection. Sporadic occurrence of other immune related peptides/proteins also pointed to a rapid response (0.5-3 h p.i.). Altogether, our results indicate that the gills of blue mussels constitute an important first line defence adapted to act at the pH of seawater. The antimicrobial activity of the gills is however modulated when mussels are under the pressure of ocean acidification, which may give future advantages for invading pathogens.

  20. Identification of a Novel Nonstructural Protein, VP9, from White Spot Syndrome Virus: Its Structure Reveals a Ferredoxin Fold with Specific Metal Binding Sites

    SciTech Connect

    Liu,Y.; Wu, J.; Song, J.; Sivaraman, J.; Hew, C.

    2006-01-01

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP9, a full-length protein of WSSV, encoded by open reading frame wsv230, was identified for the first time in the infected Penaeus monodon shrimp tissues, gill, and stomach as a novel, nonstructural protein by Western blotting, mass spectrometry, and immunoelectron microscopy. Real-time reverse transcription-PCR demonstrated that the transcription of VP9 started from the early to the late stage of WSSV infection as a major mRNA species. The structure of full-length VP9 was determined by both X-ray and nuclear magnetic resonance (NMR) techniques. It is the first structure to be reported for WSSV proteins. The crystal structure of VP9 revealed a ferredoxin fold with divalent metal ion binding sites. Cadmium sulfate was found to be essential for crystallization. The Cd2+ ions were bound between the monomer interfaces of the homodimer. Various divalent metal ions have been titrated against VP9, and their interactions were analyzed using NMR spectroscopy. The titration data indicated that VP9 binds with both Zn2+ and Cd2+. VP9 adopts a similar fold as the DNA binding domain of the papillomavirus E2 protein. Based on our present investigations, we hypothesize that VP9 might be involved in the transcriptional regulation of WSSV, a function similar to that of the E2 protein during papillomavirus infection of the host cells.

  1. CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in transendothelial migration.

    PubMed

    Imbert, Anne-Marie; Belaaloui, Ghania; Bardin, Florence; Tonnelle, Cecile; Lopez, Marc; Chabannon, Christian

    2006-10-15

    Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps, similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently, CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99, albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid, natural killer (NK), or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors.

  2. Ultrastructural observations on the hamuli of the monogenean gill parasite Cichlidogyrus halli typicus.

    PubMed

    el-Naggar, M M

    1993-09-01

    The ultrastructure of a monogenean hamulus is described for the first time. The ultrastructure of the dorsal and ventral hamuli of the monogenean gill parasite Cichlidogyrus halli typicus (Price & Kirk, 1967, 76: 137-144) Revue de Zoologie et de Botanique Africaines Paperna, 1979 (Annales du Musée Royal del'Afrique Centrale 226: 1-31) is similar but there are differences between different regions of a single hamulus. Each hamulus has three layers in the middle of the hook region, a thin outer layer, a middle layer forming the bulk of the hook and a central core made up of longitudinally oriented rods. The central core disappears in the distal region of the hook and the outer layer is absent in the proximal region of the hook. Both the shaft and the roots consist of two layers, the inner layer of the shaft is made up of longitudinally oriented parallel rods, and the corresponding layer of the roots being composed of long fibrils. The longitudinally orientated rods of the central core may provide inner strength to resist the physical strain on the hook and the outer, and possibly the middle layers, may provide chemical resistance to host's tissue secretion. The distal region of each root is covered with a fibrous hood, which appear to be a specialized region for binding muscle fibres to the sclerite.

  3. Effects of nicotine on zebrafish: A comparative response between a newly established gill cell line and whole gills.

    PubMed

    Nathiga Nambi, K S; Abdul Majeed, S; Taju, G; Sivasubbu, Sridhar; Sarath Babu, V; Sahul Hameed, A S

    2017-05-01

    A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18-22μm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish.

  4. External gill motility and striated muscle presence in the embryos of anuran amphibians.

    PubMed

    Nokhbatolfoghahai, M; Downie, J R; Atherton, L

    2013-02-01

    Anuran external gills were assessed for motility and striated muscle content in 16 species from seven families. Motility of three kinds was observed. Pulsatory movements related to heart beat rhythm were common. In embryos developing to a late stage before hatching, movements of the whole embryo were frequent, with gills rearranging as a consequence. The only clearly active movement, presumably muscle driven, was 'gill flicking', a posterior movement of the entire gill into the body either on one side only, or both together, followed by a return to the normal spread-out position. Some species may actively spread their gills when hanging from the water surface film, but we did not observe this. In some species, active gill movement developed over time, but we were not able to follow all species over such a developmental sequence. The relationship between active motility and muscle content was good in most cases. Observations on late stage embryos of the aromobatid Mannophryne trinitatis are presented for the first time. In one species, we noted spread external gills being used to adhere hatchlings to a surface.

  5. Intraspecific variation in gill morphology of juvenile Nile perch, Lates niloticus, in Lake Nabugabo, Uganda

    USGS Publications Warehouse

    Paterson, Jaclyn A.; Chapman, Lauren J.; Schofield, Pamela J.

    2010-01-01

    Several studies have demonstrated intraspecific variation in fish gill size that relates to variation in dissolved oxygen (DO) availability across habitats. In Lake Nabugabo, East Africa, ecological change over the past 12 years has coincided with a shift in the distribution of introduced Nile perch such that a larger proportion of the population now inhabits waters in or near wetland ecotones where DO is lower than in open waters of the lake. In this study, we compared gill size of juvenile Nile perch between wetland and exposed (open-water) habitats of Lake Nabugabo in 2007, as well as between Nile perch collected in 1996 and 2007. For Nile perch of Lake Nabugabo [<20 cm total length (TL)], there was a significant habitat effect on some gill traits. In general, fish from wetland habitats were characterized by a longer total gill filament length and average gill filament length than conspecifics from exposed habitats. Nile perch collected from wetland areas in 2007 had significantly larger gills (total gill filament length) than Nile perch collected in 1996, but there was no difference detected between Nile perch collected from exposed sites in 2007 and conspecifics collected in 1996.

  6. Structural changes in gill DNA reveal the effects of contaminants on Puget Sound fish.

    PubMed Central

    Malins, Donald C; Stegeman, John J; Anderson, Jack W; Johnson, Paul M; Gold, Jordan; Anderson, Katie M

    2004-01-01

    Structural differences were identified in gill DNA from two groups of English sole collected from Puget Sound, Washington, in October 2000. One group was from the industrialized Duwamish River (DR) in Seattle and the other from relatively clean Quartermaster Harbor (QMH). Chemical markers of sediment contamination [e.g., polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)] established that the DR was substantially more contaminated than QMH. The levels of these chemicals in the sediments of both sites were consistent with levels of cytochrome P450 1A (CYP1A) expression in the gills of English sole from the same sites. Structural differences in gill DNA between the groups were evinced via statistical models of Fourier transform-infrared (FT-IR) spectra. Marked structural damage was found in the gill DNA of the DR fish as reflected in differences in base functional groups (e.g., C-O and NH2) and conformational properties (e.g., arising from perturbations in vertical base stacking interactions). These DNA differences were used to discriminate between the two fish groups through principal components analysis of mean FT-IR spectra. In addition, logistic regression analysis allowed for the development of a "DNA damage index" to assess the effects of contaminants on the gill. The evidence implies that environmental chemicals contribute to the DNA changes in the gill. The damaged DNA is a promising marker for identifying, through gill biopsies, contaminant effects on fish. PMID:15064153

  7. Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

    PubMed

    Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2010-04-12

    A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability.

  8. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2011-01-01

    A primary cultured gill epithelium from the model organism Tetraodon nigroviridis (spotted green puffer fish) has been developed for the study of claudin tight junction (TJ) proteins and their potential role in the regulation of paracellular permeability across the gills of fishes. The cultured preparation is composed of polygonal epithelial cells that exhibit TJ protein immunoreactivity around the periphery and develop a surface morphology of concentric apical microridges. There is an absence of cells exhibiting intense Na+-K+-ATPase immunoreactivity and taken together, these characteristics indicate that the epithelium is composed of gill pavement cells only. In Tetraodon, 52 genes encoding for claudin isoforms (Tncldn) have been identified and 32 of these genes are expressed in whole gill tissue. Of these genes, 12 are responsive to alterations in environmental salinity in vivo (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b). All claudin isoforms found in whole gill tissue can be found in cultured pavement cell gill epithelia with the exception of Tncldn6, -10d, and -10e. The cultured preparation is suitable for studying the "molecular machinery" of TJ proteins in fish gill pavement cells.

  9. Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation.

    PubMed

    Jung, Dawoon; Sato, J Denry; Shaw, Joseph R; Stanton, Bruce A

    2012-03-01

    Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.

  10. Microbial proliferation on gill structures of juvenile European lobster ( Homarus gammarus) during a moult cycle

    NASA Astrophysics Data System (ADS)

    Middlemiss, Karen L.; Urbina, Mauricio A.; Wilson, Rod W.

    2015-12-01

    The morphology of gill-cleaning structures is not well described in European lobster ( Homarus gammarus). Furthermore, the magnitude and time scale of microbial proliferation on gill structures is unknown to date. Scanning electron microscopy was used to investigate development of setae in zoea, megalopa and juvenile stages (I-V). Microbes were classified and quantified on gill structures throughout a moult cycle from megalopa (stage IV) to juvenile (stage V). Epipodial serrulate setae, consisting of a naked proximal setal shaft with the distal portion possessing scale-like outgrowths (setules), occur only after zoea stage III. After moulting to megalopa (stage IV), gill structures were completely clean and no microbes were visible on days 1 or 5 postmoult. Microbial proliferation was first evident on day 10 postmoult, with a significant 16-fold increase from day 10 to 15. Rod-shaped bacteria were initially predominant (by day 10); however, by day 15 the microbial community was dominated by cocci-shaped bacteria. This research provides new insights into the morphology of gill-grooming structures, the timing of their development, and the magnitude, timescale and characteristics of gill microbial proliferation during a moult cycle. To some degree, the exponential growth of epibionts on gills found during a moult cycle will likely impair respiratory (gas exchange) and ion regulatory function, yet further research is needed to evaluate the physiological effects of the exponential bacterial proliferation documented here.

  11. Ultramorphological changes in gill rakers of Astyanax altiparanae (Characidae) kept in contaminated environments.

    PubMed

    Lopes, F P; Pereira, B F; Alves, R M S; Valim, J R T; Figueiredo, F A T; Pitol, D L; Caetano, F H

    2017-02-14

    Most water bodies in Brazil, and in the world, are contaminated by some types of pollutants, ranging from sewage to metal/chemicals, carcinogenic products, and biodegradable detergents. Despite the extensive knowledge on their effects on fish biology and especially on gill morphology, research that concerns their impacts on gill rakers and implications in parameters such as food consumption cannot be found in the literature. Gill rakers are vital because, together with gills, they are responsible for the defense and protection of the organism and for selecting appropriate food for survival. When detergents, which can act as toxic chemical agents, get in contact with the body of the fish, they can cause severe effects that must be understood. Therefore, our study investigated ultramorphological changes in gill rakers of Astyanax altiparanae (Lambeth) caused by the exposure to biodegradable detergents. Fish were exposed to a 1 mg/L dilution of a mixture of detergents and pure water from an artesian well for 5 months. Results revealed that the first month of exposure to detergent caused dilation of chemical receptors in taste buds and the rise of a large number of orifices for mucus release among pavement cells in gill rakers, although only a small amount of mucus was found in fish exposed both to pure water and the detergent dilution. After 5 months, there was an increase in the dilation of these chemoreceptors, excess mucus on gill rakers of detergent groups, and the emergence of microbridges between microridges in pavement cells.

  12. Gill erosion and herpesvirus in Crassostrea gigas cultured in Baja California, Mexico.

    PubMed

    Vásquez-Yeomans, Rebeca; García-Ortega, Mauricio; Cáceres-Martínez, Jorge

    2010-03-09

    Recurrent episodes of mortality of Crassostrea gigas cultured in northwestern Mexico have been occurring since 1997. Previous studies on bacteria, protozoans, and metazoans as presumptive causal agents have been inconclusive. However, erosions in the marginal indentation of gills have been frequently observed in oysters from areas affected by mortality events, and in 2000 those lesions were associated with the detection of a herpes-like virus. The present study aimed to describe the histological alterations of eroded gills and to determine whether ostreid herpesvirus 1 (OsHV-1) or a related virus was associated with them using in situ hybridization (ISH). Histology showed that gill filaments were fused. In severe cases, deformation of the interlamellar junctions, swelling, and the loss of water channels was observed. ISH analysis revealed the presence of OsHV-1 DNA or a related virus in cells of the gills. Some labeled cells were large with dark granules inside their cytoplasm. These cells were surrounded by infiltrating hemocytes. Some cells interpreted as hemocytes were labeled and observed in eroded and non-eroded areas of the gill. Large cells detected by ISH were also observed by conventional histology with hematoxylin-eosin staining. Whether the virus produces the erosions in the gills, or the erosions in the gills are produced by an unknown condition and favor the presence of the virus, remains unresolved. It is also not clear whether the lesions contribute to mortality.

  13. Temporal variation in the antioxidant defence system and lipid peroxidation in the gills and mantle of hydrothermal vent mussel Bathymodiolus azoricus

    NASA Astrophysics Data System (ADS)

    Company, Rui; Serafim, Angela; Cosson, Richard; Fiala-Médioni, Aline; Dixon, David; João Bebianno, Maria

    2006-07-01

    Hydrothermal vent mussels are exposed continually to toxic compounds, including high metal concentrations and other substances like dissolved sulphide, methane and natural radioactivity. Fluctuations in these parameters appear to be common because of the characteristic instability of the hydrothermal environment. Temporal variation in the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidases (Total GPx), selenium dependent glutathione peroxidases (Se-GPx)), metallothioneins and lipid peroxidation (LPO) in the gills and mantle of the mussel Bathymodiolus azoricus from Menez-Gwen hydrothermal vent site was evaluated and related to the accumulated metal concentrations (Ag, Cu, Cd, Fe, Mn and Zn) in the tissues. Maximum antioxidant enzyme activities in the gills were detected in the beginning of summer, followed by a gradual decrease throughout the following months. One year after, the levels of antioxidant enzyme activities were similar to those reported one year before. LPO in this tissue exhibited a similar temporal variation trend. A different pattern of temporal variation in antioxidant enzyme activities was observed in the mantle, with a gradual increase from summer to the end of autumn (November). LPO in the mantle exhibited an almost reverse trend of temporal variation to that of antioxidant enzyme activities in this tissue. Antioxidant defences in the gills of B. azoricus were significantly enhanced with increasing concentrations of Ag, Cu and Mn, while negative relationships between antioxidant enzymes and Cd, Cu, Mn and Zn concentrations in the mantle were observed, suggesting different pathways of reactive oxygen species (ROS) production and that these tissues responded differently to the metal accumulation. However, temporal variation in biomarkers of defence and damage were in general similar to coastal bivalve species and can be associated with temporal variations of the physiological status due to reproduction

  14. Evolution of the branchiostegal membrane and restricted gill openings in Actinopterygian fishes.

    PubMed

    Farina, Stacy C; Near, Thomas J; Bemis, William E

    2015-06-01

    A phylogenetic survey is a powerful approach for investigating the evolutionary history of a morphological characteristic that has evolved numerous times without obvious functional implications. Restricted gill openings, an extreme modification of the branchiostegal membrane, are an example of such a characteristic. We examine the evolution of branchiostegal membrane morphology and highlight convergent evolution of restricted gill openings. We surveyed specimens from 433 families of actinopterygians for branchiostegal membrane morphology and measured head and body dimensions. We inferred a relaxed molecular clock phylogeny with branch length estimates based on nine nuclear genes sampled from 285 species that include all major lineages of Actinopterygii. We calculated marginal state reconstructions of four branchiostegal membrane conditions and found that restricted gill openings have evolved independently in at least 11 major actinopterygian clades, and the total number of independent origins of the trait is likely much higher. A principal component analysis revealed that fishes with restricted gill openings occupy a larger morphospace, as defined by our linear measurements, than do fishes with nonrestricted openings. We used a decision tree analysis of ecological data to determine if restricted gill openings are linked to certain environments. We found that fishes with restricted gill openings repeatedly occur under a variety of ecological conditions, although they are rare in open-ocean pelagic environments. We also tested seven ratios for their utility in distinguishing between fishes with and without restricted gill openings, and we propose a simple metric for quantifying restricted gill openings (RGO), defined as a ratio of the distance from the ventral midline to the gill opening relative to half the circumference of the head. Functional explanations for this specialized morphology likely differ within each clade, but its repeated evolution indicates a need

  15. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer.

    PubMed

    Chen, Wei-Ching; Chang, Yung-Sheng; Hsu, Hui-Ping; Yen, Meng-Chi; Huang, Hau-Lun; Cho, Chien-Yu; Wang, Chih-Yang; Weng, Tzu-Yang; Lai, Po-Ting; Chen, Ching-Shih; Lin, Yih-Jyh; Lai, Ming-Derg

    2015-12-15

    CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

  16. Gill ectoparasites of Barbus martorelli (Teleostean: Cyprinidae) from a tropical watercourse (Cameroon, Africa): conflict or coexistence?

    PubMed

    Tombi, J; Bilong Bilong, C F; Morand, S

    2011-02-01

    The structure and stability of parasite communities have been mainly explained by high diversity and strong interactions among parasite species. During 16 months, 558 Barbus martorelli gill infracommunities were studied in a tropical zone to determine whether parasite infrapopulations interact. Three levels were retained: the infracommunity level, the gill filament level, and the filament fraction level. Single species infections in Barbus martorelli were very rare and only concerned the core species: Dactylogyrus bopeleti, D. insolitus, D. simplex and Myxobolus barbi. Mixed infections appeared as a general rule in this fish species. Interspecific interactions at all three levels were statistically non significant. Our results suggest that Barbus martorelli gill parasites are non interactive (isolationist).

  17. CD Rainbows

    ERIC Educational Resources Information Center

    Ouseph, P. J.

    2007-01-01

    Several papers have been published on the use of a CD as a grating for undergraduate laboratories and/or for high school and college class demonstrations. Four years ago "The Physics Teacher" had a spectacular cover picture showing emission spectrum as viewed through a CD with no coating. That picture gave the impetus to develop a system that can…

  18. Effects of heavy metals on the Ca(2+)-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.).

    PubMed

    Viarengo, A; Mancinelli, G; Pertica, M; Fabbri, R; Orunesu, M

    1993-11-01

    1. Heavy metals (Hg2+, Cu2+, Cd2+, Zn2+, Pb2+) at micromolar concentrations strongly inhibit the Ca(2+)-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate. 2. All the heavy metals tested inhibit the Ca(2+)-ATPase activity, the effect following the order: Hg2+ > Pb2+ > Cu2+ > Cd2+ > Zn2+; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals. 3. The inhibitory effects of Cu2+ on Ca(2+)-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 microM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.

  19. A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells

    PubMed Central

    Reusch, Uwe; Burkhardt, Carmen; Fucek, Ivica; Le Gall, Fabrice; Le Gall, Mikaelle; Hoffmann, Karin; Knackmuss, Stefan HJ; Kiprijanov, Sergej; Little, Melvyn; Zhukovsky, Eugene A

    2014-01-01

    To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced koff, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30+ target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin’s lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells. PMID:24670809

  20. Artificial gills for robots: MFC behaviour in water.

    PubMed

    Ieropoulos, Ioannis; Melhuish, Chris; Greenman, John

    2007-09-01

    This paper reports on the first stage in developing microbial fuel cells (MFCs) which can operate underwater by utilizing dissolved oxygen. In this context, the cathodic half-cell is likened to an artificial gill. Such an underwater power generator has obvious potential for autonomous underwater robots. The electrical power from these devices increased proportionately with water flow rate, temperature and salinity. The current output at ambient temperature (null condition) was 32 microA and this increased by 200% (approximately 100 microA) as a result of a corresponding temperature increase (DeltaT) of 52 degrees C. Similarly, the effect of increasing the water flow rate resulted in an increase in the MFC output ranging from 135% to 150%. Furthermore, the same positive effect was recorded when artificial seawater was used instead, in which case the increase in the MFC current output was >100% (from 32 to 65 microA). There was a distinct difference in the MFC performance when operated under low turbulent as opposed to high turbulent flow rates. These findings can be advantageous in the design of underwater autonomous robots.

  1. Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and nectin-2 (PRR-2/CD112).

    PubMed

    Tahara-Hanaoka, Satoko; Shibuya, Kazuko; Onoda, Yuko; Zhang, Hua; Yamazaki, Satoshi; Miyamoto, Akitomo; Honda, Shin-Ichiro; Lanier, Lewis L; Shibuya, Akira

    2004-04-01

    CD226 (DNAM-1) is an adhesion molecule involved in NK and T cell-mediated cytotoxicity against certain tumors. Here, we have identified the human poliovirus receptor-related (PRR) family members CD155 [poliovirus receptor (PVR)] and CD112 (nectin-2/PRR-2) as the ligands for human CD226. Ectopic expression of human CD155 and/or CD112 rendered mouse BW5147 T cells more susceptible to IL-2-activated T and NK cell-mediated cytotoxicity, and killing was specifically inhibited by anti-CD226 mAb, demonstrating functional interactions of CD226 with CD155 and CD112. Although the binding affinities between soluble CD226 and CD155 or CD112 were comparable, the homophilic interaction of cell-surface CD112 may adversely affect CD226 binding to CD112. We also demonstrate that ligation of CD226 and LFA-1 with their respective ligands cooperates in triggering cytotoxicity and cytokine secretion by T and NK cells.

  2. Gilles de la Tourette Syndrome: A Review and Implications for Educators.

    ERIC Educational Resources Information Center

    Lemons, Laurie A.; Barber, William H.

    1991-01-01

    Gilles de la Tourette syndrome is a disorder characterized by multiple involuntary motor and verbal tics. This review covers the history, symptoms, diagnostic criteria, past and present treatments, associated disorders, and various educational techniques. (Author/DB)

  3. The Gills Creek Watershed Association of Columbia, SC awarded $30,000 Environmental Justice Small Grant

    EPA Pesticide Factsheets

    ATLANTA - An Environmental Justice Small Grant from the U.S. Environmental Protection Agency (EPA) has been awarded to the Gills Creek Watershed Association. for their project titled: Exposure to mercury through subsistence fishing: Assessment and o

  4. Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

    PubMed

    Anjos, V A; Galvão, J S; Santos, V R S; Souza, M M

    2016-11-01

    Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

  5. The fish gill: site of action and model for toxic effects of environmental pollutants.

    PubMed Central

    Evans, D H

    1987-01-01

    The gill epithelium is the site of gas exchange, ionic regulation, acid-base balance, and nitrogenous waste excretion by fishes. The last three processes are controlled by passive and active transport of various solutes across the epithelium. Various environmental pollutants (e.g., heavy metals, acid rain, and organic xenobiotics) have been found to affect the morphology of the gill epithelium. Associated with these morphological pathologies, one finds alterations in blood ionic levels, as well as gill Na,K-activated ATPase activity and ionic fluxes. Such physiological disturbances may underly the toxicities of these pollutants. In addition, the epithelial transport steps which are affected in the fish gill model resemble those described in the human gut and kidney, sites of action of a variety of environmental toxins. Images FIGURE 1. a FIGURE 1. b FIGURE 3. PMID:3297663

  6. Three Cases of Palatal Tics and Gilles De La Tourette Syndrome.

    PubMed

    Rizzo, Renata; Cath, Danielle; Pavone, Piero; Tijssen, Marina; Robertson, Mary M

    2015-08-01

    Five patients with palatal tics and Gilles de la Tourette syndrome have been previously reported. Little is known about the characteristics of palatal tics given that there are so few reports. On one hand, palatal tics may be rare. Alternatively, they may be less well recognized than repetitive eye blinking or sniffing, which are both obvious and, therefore, more often reported. We describe 3 patients with palatal tics and Gilles de la Tourette syndrome. We also review the 5 patients reported in the literature and explore whether there are characteristic features among this group of 8 cases. The 8 patients had the following features: (1) Personal history of other multiple motor/vocal tics, (2) the presence of typical Gilles de la Tourette syndrome comorbidities, (3) positive family history of tics and/or Gilles de la Tourette syndrome comorbidities, (4) the presence of audible "ear clicks," (5) younger age at onset (2 years). We suggest that palatal tics are underreported.

  7. Cadmium affects the mitochondrial viability and the acid soluble thiols concentration in liver, kidney, heart and gills of Ancistrus brevifilis (Eigenmann, 1920)

    PubMed Central

    Velasquez-Vottelerd, P.; Anton, Y.; Salazar-Lugo, R.

    2015-01-01

    The freshwater fish Ancistrus brevifilis, which is found in Venezuelan rivers, is considered a potential sentinel fish in ecotoxicological studies. The cadmium (Cd) effect on the mitochondrial viability (MV) and acid soluble thiols levels (AST) in A. brevifilis tissues (liver, kidney, heart, and gill) was evaluated. Forty-two fish with similar sizes and weights were randomly selected, of which 7 fish (with their respective replicate) were exposed for 7 and 30 days to a Cd sublethal concentration (0.1 mg.l-1). We determined the MV through a Janus Green B colorimetric assay and we obtained the concentration of AST by Ellman’s method. Mitochondrial viability decreased in fish exposed to Cd for 30 days with the liver being the most affected tissue. We also detected a significant decrease in AST levels was in fishes exposed to Cd for 7 days in liver and kidney tissues; these results suggests that AST levels are elevated in some tissues may act as cytoprotective and adaptive alternative mechanism related to the ROS detoxification, maintenance redox status and mitochondrial viability. Organ-specifics variations were observed in both assays. We conclude that the Cd exposure effect on AST levels and MV, vary across fish tissues and is related to the exposure duration, the molecule dynamics in different tissues, the organism and environmental conditions. PMID:26623384

  8. Life science experiments during parabolic flight: The McGill experience

    NASA Technical Reports Server (NTRS)

    Watt, D. G. D.

    1988-01-01

    Over the past twelve years, members of the Aerospace Medical Research Unit of McGill University have carried out a wide variety of tests and experiments in the weightless condition created by parabolic flight. This paper discusses the pros and cons of that environment for the life scientist, and uses examples from the McGill program of the types of activities which can be carried out in a transport aircraft such as the NASA KC-135.

  9. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    SciTech Connect

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade

  10. Trophic ecology and gill raker morphology of seven catostomid species in Iowa rivers

    USGS Publications Warehouse

    Spiegel, J.R.; Quist, M.C.; Morris, J.E.

    2011-01-01

    Understanding the trophic ecology of closely-related species is important for providing insight on inter-specific competition and resource partitioning. Although catostomids often dominate fish assemblages in lotic systems, little research has been conducted on their ecology. This study was developed to provide information on the trophic ecology of catostomids in several Iowa rivers. Food habits, diet overlap, and gill raker morphology were examined for highfin carpsucker Carpiodes velifer, quillback C.??cyprinus, river carpsucker C.??carpio, golden redhorse Moxostoma erythrurum, shorthead redhorse M.??macrolepidotum, silver redhorse M.??anisurum, and northern hogsucker Hypentelium nigricans sampled from four Iowa rivers (2009). Diet overlap among all species was calculated with Morista's index (C). Food habit niche width was quantified with Levin's index (B) and similarity in gill raker morphology was compared with analysis of covariance. Values from Morista's index suggested significant overlap in the diets of highfin carpsucker and river carpsucker (C=0.81), quillback and river carpsucker (C=0.66), and shorthead redhorse and silver redhorse (C=0.67). Levin's index indicated that golden redhorse (B=0.32), quillback (B=0.53), and river carpsucker (B=0.41) had the most generalized feeding strategies as their food niche widths were substantially wider than the other species. Gill raker length and spacing were positively correlated with the standard length of the fish for all species (gill raker length: r2=0.67-0.88, P???0.01; gill raker spacing: r2=0.63-0.73, P???0.01). Slopes of regression of gill raker length and spacing to standard lengths were significantly (P???0.05) different among species, indicating that rates of change in gill raker morphology with body length varied among species. Differences in gill raker morphology likely allow catostomids to partition resources and reduce competitive interactions. ?? 2011.

  11. Ab Initio Study on Atomic Structures and Physical Properties of CdSe Quantum Nanodots

    DTIC Science & Technology

    2009-11-25

    CdSe quantum dots , with magic number (( CdSe )13, ( CdSe )19, ( CdSe )33 and ( CdSe )34 ). Effects of organic ligand binding on the stability of CdSe as well...calculations of optical absorption spectra for CdSe quantum dots , with magic number (( CdSe )13, ( CdSe )19, ( CdSe )33 and ( CdSe )34 ), have been calculated in...1 AOARD-08-4037 Title of Proposed Project: Ab initio study on atomic structures and physical

  12. Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

    PubMed

    Meza-Romero, Roberto; Benedek, Gil; Leng, Lin; Bucala, Richard; Vandenbark, Arthur A

    2016-04-01

    Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

  13. Identification of Methanotrophic Biomarker Lipids in the Symbiont-Containing Gills of Seep Mussels

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Zahiralis, K. D.; Klein, H. P.; Morrison, David (Technical Monitor)

    1994-01-01

    Mussels collected from hydrocarbon seeps in the Gulf of Mexico grow with methane as sole carbon and energy source due to a symbiotic association with methane-oxidizing bacteria. Transmission electron micrographs of mussel gills show cells with stacked intracytoplasmic membranes similar to type I methanotrophic bacteria. Methanotrophs are known to synthesize several types of cyclic triterpenes, hopanoids and methyl sterols, as well as unique monounsaturated fatty acid, double bond positional isomers that serve as biomarkers for this group. Lipid analysis of dissected mussels demonstrated the presence of these biomarkers predominantly in the gill tissue with much smaller amounts in mantle and remaining body tissues. Gill tissue contained 1150 micrograms/g dry wt. of hopanepolyol derivatives and diplopterol while the mantle tissue contained only 17 micrograms/g. The C16 monounsaturated fatty acids (16:1) characteristic of type I methanotrophic membranes dominated the gill tissue making up 53% of the total while only 5% 16:1 was present in the mantle tissue. The methyl sterol distribution was more dispersed. The predominant sterol in all tissues was cholesterol with lesser amounts of other desmethyl and 4-methyl sterols. The gill and mantle tissues contained 3461 micrograms (17% methyl) and 2750 micrograms (5% methyl) sterol per gm dry wt., respectively. Methyl sterol accounted for 44% of the sterol esters isolated from the gill, suggesting active demethylation of the methanotrophic sterols in this tissue. The use of lipid biomarkers could provide an effective means for identifying host-symbiont relationships.

  14. Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

    PubMed

    Jarial, M S; Wilkins, J H

    2003-10-01

    The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

  15. Impact of environmental DDT concentrations on gill adaptation to increased salinity in the tilapia Sarotherodon melanotheron.

    PubMed

    Riou, Virginie; Ndiaye, Awa; Budzinski, Hélène; Dugué, Rémi; Le Ménach, Karyn; Combes, Yan; Bossus, Maryline; Durand, Jean-Dominique; Charmantier, Guy; Lorin-Nebel, Catherine

    2012-06-01

    Estuaries of tropical developing countries suffering from severe droughts induced by climate change are habitats to fish, which face drastic salinity variations and the contact with pollutants. The Western Africa tilapia Sarotherodon melanotheron is highly resistant to hypersalinity, but the effect of human-released xenobiotics on its adaptation is barely known. Controlled experiments were conducted to observe S. melanotheron gill adaptation to abrupt salinity variations in the presence of waterborne DDT, at concentrations detected in their natural habitat. The gills appeared as an important site of DDT conversion to DDD and/or depuration. A 12-days DDT exposure resulted in decreased gill epithelium thickness at all salinities (from fresh- to hypersaline-water), and the structure of gills from freshwater fish was particularly altered, relative to controls. No unbalance in tilapia blood osmolality was observed following DDT exposure, which however caused a decrease in branchial Na(+)-K(+)-ATPase (NKA) activity. Gill cellular NKA expression was reduced in salt-water, together with the expression of the CFTR chloride channel in hypersaline water. Although S. melanotheron seems very resistant (especially in seawater) to short-term waterborne DDT contamination, the resulting alterations of the gill tissue, cells and enzymes might affect longer term respiration, toxicant depuration and/or osmoregulation in highly fluctuating salinities.

  16. First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus.

    PubMed

    Jeong, Ji-Min; Kim, Ju-Won; Kim, Do-Hyung; Park, Chan-Il

    2015-04-01

    CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

  17. CD Rom.

    PubMed

    1996-02-01

    A new CD-Rom has been launched by Guy's and St Thomas' Trust's poisonous unit to help health professionals discover which species have been involved in cases of plant poisoning. The unit says thousands of people are poisoned every year by eating or touching plants - the majority of those people affected being under the age of seven. The CD-Rom covers several thousand species of plant, and has been jointly researched with Kew Gardens.

  18. CD147 in cardiovascular disease and thrombosis.

    PubMed

    Pennings, Gabrielle J; Kritharides, Leonard

    2014-10-01

    Thrombotic and inflammatory pathways play a key role in coronary artery disease (CAD) development. Extracellular matrix metalloproteinase (aka CD147) is a member of the immunoglobulin superfamily that is expressed on many cell types including hematopoietic, endothelial cells, leukocytes, keratinocytes, platelets, and others. The binding partners of CD147 are numerous and diverse, and give some indication to the various roles that CD147 can play; these include homophilic interactions, integrins, cyclophilins, glycoprotein VI (GPVI), caveolin 1, and monocarboxylate transporters. Recent evidence suggests a role for CD147 in both thrombosis and inflammation, as well as involvement in CAD and cancer. In this review, we summarize the role of CD147 and its binding partners in platelets, thrombosis, and arterial disease and assess mechanistic aspects of CD147 biology.

  19. Gilles Deleuze: psychiatry, subjectivity, and the passive synthesis of time.

    PubMed

    Roberts, Marc

    2006-10-01

    Abstract Although 'modern' mental health care comprises a variety of theoretical approaches and practices, the supposed identification of 'mental illness' can be understood as being made on the basis of a specific conception of subjectivity that is characteristic of 'modernity'. This is to say that any perceived 'deviation' from this characteristically 'modern self' is seen as a possible 'sign' of 'mental illness', given a 'negative determination', and conceptualized in terms of a 'deficiency' or a 'lack'; accordingly, the 'ideal''therapeutic' aim of 'modern' mental health care can be understood as the 'rectification' of that 'deficiency' through a 're-instatement' of the 'modern self'. Although contemporary mental health care is increasingly becoming influenced by the so-called 'death' of the 'modern self', this paper will suggest that it is the work of the 20th century French philosopher, Gilles Deleuze, that is able to provide mental health care with a coherent determination of a 'post-modern self'. However, a Deleuzian account of subjectivity stands in stark contrast to 'modernity's' conception of subjectivity and, as such, this paper will attempt to show how this 'post-modern' subjectivity challenges many of the assumptions of 'modern' mental health care. Moreover, acknowledging the complexity and the perceived difficulty of Deleuze's work, this paper will provide an account of subjectivity that can be understood as 'Deleuzian' in its orientation, rather than 'Deleuze's theory of subjectivity', and therefore, this paper also seeks to stimulate further research and discussion of Deleuze's work on subjectivity, and how that work may be able to inform, and possibly even reform, the theoretical foundations and associated diagnostic and therapeutic practices of psychiatry, psychotherapy, and mental health nursing.

  20. Genome scan for linkage to Gilles de la Tourette syndrome

    SciTech Connect

    Barr, C.L.; Livingston, J.; Williamson, R.

    1994-09-01

    Gilles de la Tourette Syndrome (TS) is a familial, neuropsychiatric disorder characterized by chronic, intermittent motor and vocal tics. In addition to tics, affected individuals frequently display symptoms such as attention-deficit hyperactivity disorder and/or obsessive compulsive disorder. Genetic analyses of family data have suggested that susceptibility to the disorder is most likely due to a single genetic locus with a dominant mode of transmission and reduced penetrance. In the search for genetic linkage for TS, we have collected well-characterized pedigrees with multiple affected individuals on whom extensive diagnostic evaluations have been done. The first stage of our study is to scan the genome systematically using a panel of uniformly spaced (10 to 20 cM), highly polymorphic, microsatellite markers on 5 families segregating TS. To date, 290 markers have been typed and 3,660 non-overlapping cM of the genome have been excluded for possible linkage under the assumption of genetic homogeneity. Because of the possibility of locus heterogeneity overall summed exclusion is not considered tantamount to absolute exclusion of a disease locus in that region. The results from each family are carefully evaluated and a positive lod score in a single family is followed up by typing closely linked markers. Linkage to TS was examined by two-point analysis using the following genetic model: single autosomal dominant gene with gene frequency .003 and maximum penetrance of .99. An age-of-onset correction is included using a linear function increasing from age 2 years to 21 years. A small rate of phenocopies is also incorporated into the model. Only individuals with TS or CMT according to DSM III-R criteria were regarded as affected for the purposes of this summary. Additional markers are being tested to provide coverage at 5 cM intervals. Moreover, we are currently analyzing the data non-parametrically using the Affected-Pedigree-Member Method of linkage analysis.

  1. Oxidative stress response in gill and liver of Liza saliens, from the Esmoriz-Paramos coastal lagoon, Portugal.

    PubMed

    Fernandes, C; Fontaínhas-Fernandes, A; Ferreira, M; Salgado, M A

    2008-08-01

    Tissue-specific responses against oxidative stress and lipid peroxidation were analyzed in wild adult mullet (Liza saliens) caught in the Portuguese coastal lagoon Esmoriz-Paramos. Parameters measured were catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities in liver and gill tissues and lipid peroxidation. The enzyme activities were related to gill histopathological alterations, as well as to heavy metals (Cu and Zn) concentrations in these tissues. Gill epithelium of L. saliens showed histological alterations, such as epithelial hyperplasia resulting in lamellar fusion, epithelial lifting, vasodilatation, and lamellar aneurisms, with a prevalence ranging from 62% to 92%. The highest Cu content was found in liver (379 mg x kg(-1)), while the highest Zn content was observed in gill (119 mg x kg(-1)). SOD and CAT activities showed differences between gill and liver. The highest activities found were SOD in gill (10.1 U/mg protein) and CAT in liver (39.2 mmol/min/mg protein). In gill, CAT activity was negatively related to both Cu levels and gill lifting, while a positive relationship was found between SOD activity and fish age. The positive relationship between Cu and CAT activity in liver suggests that an increase in metabolic level is related to Cu-induced oxidative stress. The decrease in gill CAT activity can be due to osmotic stress caused by damaged gill epithelium. CAT activity in liver is an appropriate biomarker of oxidative stress in the Esmoriz-Paramos lagoon.

  2. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4.

    PubMed

    Suetake, Hiroaki; Araki, Kyosuke; Suzuki, Yuzuru

    2004-08-01

    We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15-20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.

  3. Structures of CD6 and Its Ligand CD166 Give Insight into Their Interaction

    PubMed Central

    Chappell, Paul E.; Garner, Lee I.; Yan, Jun; Metcalfe, Clive; Hatherley, Deborah; Johnson, Steven; Robinson, Carol V.; Lea, Susan M.; Brown, Marion H.

    2015-01-01

    Summary CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents. PMID:26146185

  4. The CD1 size problem: lipid antigens, ligands, and scaffolds

    PubMed Central

    Ly, Dalam

    2014-01-01

    Whereas research on CD1d has emphasized a few glycosyl ceramides, the broader family of four human CD1 antigen-presenting molecules binds hundreds of distinct self-lipids. Individual lipid types bind within CD1 grooves in different ways, such that they partially fill the groove, match the groove volume, or protrude substantially from the groove. These differing modes of binding can now be connected to differing immunological functions, as individual lipids can act as stimulatory antigens, inhibitory ligands, or space-filling scaffolds. Because each type of CD1 protein folds to produce antigen-binding grooves with differing sizes and shapes, CD1a, CD1b, CD1c, CD1d, and CD1e have distinct mechanisms of capturing self-lipids and exchanging them for foreign lipids. The size discrepancy between endogeneous lipids and groove volume is most pronounced for CD1b. Recent studies show that the large CD1b cavity can simultaneously bind two self-lipids, the antigen, and its scaffold lipid, which can be exchanged for one large bacterial lipid. In this review, we will highlight recent studies showing how cells regulate lipid antigen loading and the roles CD1 groove structures have in control of the presentation of chemically diverse lipids to T cells. PMID:24658584

  5. CD1A — EDRN Public Portal

    Cancer.gov

    CD1A is a transmembrane glycoprotein that is structurally related to the major histocompatibility complex (MHC) proteins and forms heterodimers with beta-2-microglobulin. CD1A functions as an antigen-presenting protein that binds self and non-self lipid and glycolipid antigens and presents them to T-cell receptors on natural killer T-cells.

  6. Alterations in gill structure in tropical reef fishes as a result of elevated temperatures

    PubMed Central

    Bowden, A.J.; Gardiner, N.M.; Couturier, C.S.; Stecyk, J.A.W.; Nilsson, G.E.; Munday, P.L.; Rummer, J.L.

    2015-01-01

    Tropical regions are expected to be some of the most affected by rising sea surface temperatures (SSTs) because seasonal temperature variations are minimal. As temperatures rise, less oxygen dissolves in water, but metabolic requirements of fish and thus, the demand for effective oxygen uptake, increases. Gill remodelling is an acclimation strategy well documented in freshwater cyprinids experiencing large seasonal variations in temperature and oxygen as well as an amphibious killifish upon air exposure. However, no study has investigated whether tropical reef fishes remodel their gills to allow for increased oxygen demands at elevated temperatures. We tested for gill remodelling in five coral reef species (Acanthochromis polyacanthus, Chromis atripectoralis, Pomacentrus moluccensis, Dascyllus melanurus and Cheilodipterus quinquelineatus) from populations in northern Papua New Guinea (2° 35.765′ S; 150° 46.193′ E). Fishes were acclimated for 12-14 days to 29 and 31 °C, encompassing their seasonal range (29-31 °C), and 33 and 34 °C to account for end-of-century predicted temperatures. We measured lamellar perimeter, cross-sectional area, base thickness, and length for five filaments on the 2nd gill arches and qualitatively assessed 3rd gill arches via scanning electron microscopy (SEM). All species exhibited significant differences in the quantitative measurements made on the lamellae, but no consistent trends with temperature were observed. SEM only revealed alterations in gill morphology in P. moluccensis. The overall lack of changes in gill morphology with increasing temperature suggests that these near-equatorial reef fishes may fail to maintain adequate O2 uptake under future climate scenarios unless other adaptive mechanisms are employed. PMID:24862962

  7. Gill Morphology and Oxygen Diffusion Distance in Juvenile Striped Killifish, Fundulus majalis

    NASA Astrophysics Data System (ADS)

    McEnroe, M.; Rivera, L.; La Fortune, B.; Miller, A.

    2010-12-01

    Striped killifish (Fundulus majalis) are an important estuarine forage fish. Larvae and juveniles utilize shallow marsh pools which become warm (>30 C) and hypoxic (DO < 2mg/l) in summer. To investigate potential morphological adaptations to this environment we studied gill morphology. Gill surface area (GSA) and oxygen diffusion distance are important parameters for oxygen uptake, as expressed by Fick's Equation for diffusion. To measure these parameters fish (N=20, 20-50 mm TL) were collected from marsh pools and adjacent Long Island Sound in late summer, weighed, and measured. The gills were fixed in Karnovsky’s solution. For scanning electron microscopy (SEM) they were rinsed, dehydrated in a graded ETOH series, and critically point dried, sputter-coated and were observed with ISR-SR-50 SEM. Gill morphology (number of filaments, filament length, lamellar density, and lamellar size) were quantified using SEM. Total gill surface area (GSA) was calculated using the method of Hughes (1984); GSA = L* n* bl where L = sum of filament lengths, n = number of lamellae/mm, and bl = bilateral lamellar surface area. To measure oxygen diffusion distance from water to blood, samples were embedded in Araldite 502/Embed 812 TM plastic medium for transmission electron microscopy (TEM). Stained (uranyl acetate and calcinated lead citrate) thin sections were examined using a FEI/Philips Morgagni 268 transmission electron microscope (TEM). Gill lamellar diffusion distance was measured using the technique of Matey et al., 2008 and found to be low (1.0 µm ± 0.4; mean ± SD; N= 17). Gill structure and oxygen diffusion distance will be compared to other fishes from normoxic and hypoxic environments.

  8. Quantitative molecular phenotyping of gill remodeling in a cichlid fish responding to salinity stress.

    PubMed

    Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

    2013-12-01

    A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into

  9. Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander, Hynobius nigrescens Stejneger.

    PubMed

    Uchiyama, Minoru; Kumano, Tomoko; Komiyama, Makiko; Yoshizawa, Hideki; Matsuda, Kouhei

    2011-08-01

    In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na(+) , K(+) -ATPase (NKA), vacuolar-type H(+) -ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43-65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria-rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA-, NKA- and ENaC-, and VHA-positive cells. VHA-immunoreactive cells as well as NKA-positive cells were observed during IY stages 43-65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid-base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium.

  10. Tissue distribution of CD6 and CD6 ligand in cattle: expression of the CD6 ligand (CD166) in the autonomic nervous system of cattle and the human.

    PubMed

    Konno, A; Ahn, J S; Kitamura, H; Hamilton, M J; Gebe, J A; Aruffo, A; Davis, W C

    2001-06-01

    We studied the tissue distribution of CD6(+) lymphocytes and cells expressing the CD6 ligand (also known as activated leukocyte cell adhesion molecule CD166) in calves by immunohistochemistry using an anti-bovine CD6 monoclonal antibody (mAb), a human CD6 (huCD6)-immunoglobulin G1 fusion protein (huCD6-Ig), and an anti-human CD166 (anti-huCD166) mAb. The huCD6-Ig and anti-huCD166 mAb bound to the sympathetic and parasympathetic nerve fibers but not to myelinated nerve fibers in the spinal nerve. Studies with human tissue using the anti-huCD166 mAb yielded identical patterns of labeling. Dense accumulations of CD6(+) lymphocytes were present in areas of the thymuses and spleens of calves, in areas innervated by huCD6-Ig(+) nerves. The cDNAs encoding the bovine CD166 and CD6 were isolated from the sympathetic ganglion and spleen, respectively. Predicted amino acid residues that are important for human and mouse CD6-CD166 binding were also conserved in bovine CD6 and CD166. Bovine CD166 transcripts were detected by reverse transcriptase-PCR in all the tissues that bound huCD6-Ig. These results show that the bovine orthologue of CD166 was constitutively expressed in the autonomic nervous systems of cattle and suggest that CD6(+) lymphocytes adhere to CD166(+) autonomic nerve terminals via CD6.

  11. Use of polymer-bound Schiff base as a new liquid binding agent of diffusive gradients in thin-films for the measurement of labile Cu²⁺, Cd²⁺ and Pb²⁺.

    PubMed

    Fan, Hong-Tao; Liu, Jin-Xiu; Sui, Dian-Peng; Yao, Hui; Yan, Feng; Sun, Ting

    2013-09-15

    A new diffusive gradients in thin-films (DGT) device with solution of polymer-bound Schiff base (Py-PEI) derived from poly(ethyleneimine) and 2-pyridinecarboxaldehyde as the binding agent (Py-PEI DGT) was developed for measuring labile Cu(2+), Cd(2+) and Pb(2+) in waters. In synthetic solution, uptake percentages of Cu(2+), Cd(2+) and Pb(2+) by Py-PEI DGT were 99.2 ± 4.4%, 103.7 ± 4.8% and 98.7 ± 5.5% of the total metals, respectively. Performance of Py-PEI DGT was independent of pH 4-8.5 and ionic strength 1 × 10(-4)-0.1 mol L(-1). The uptake of labile metals showed agreement with the theoretical values of free ions in EDTA solution, and decreased with increase of humic acid (HA) and dissolved organic carbon (DOC) contents in water due to the complexation of HA and DOC with metals. DGT devices containing different liquid binding agents (Py-PEI, polyacrylate (PA) and poly(4-styrenesulfonate) (PSS)) were deployed in the same solution. The higher uptake percentages of Py-PEI DGT (13.2 ± 1.1, 26.5 ± 2.4 and 62.7 ± 3.2% of total Cu(2+), Cd(2+) and Pb(2+)) were obtained with respective to PSS DGT (6.7 ± 0.6, 9.2 ± 0.9 and 29.6 ± 2.7% of total above metals) and PA DGT (8.9 ± 0.9 and 16.2 ± 1.1% of total Cu(2+) and Cd(2+)) due to its relatively strong binding affinity with metals.

  12. Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b.

    PubMed

    Choi, Dong W; Do, Young S; Zea, Corbin J; McEllistrem, Marcus T; Lee, Sung-W; Semrau, Jeremy D; Pohl, Nicola L; Kisting, Clint J; Scardino, Lori L; Hartsel, Scott C; Boyd, Eric S; Geesey, Gill G; Riedel, Theran P; Shafe, Peter H; Kranski, Kim A; Tritsch, John R; Antholine, William E; DiSpirito, Alan A

    2006-12-01

    Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV-visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.

  13. Sequencing and expression analysis of CD3γ/δ and CD3ɛ chains in mandarin fish, Siniperca chuatsi

    NASA Astrophysics Data System (ADS)

    Guo, Zheng; Nie, Pin

    2013-01-01

    The genomic and cDNA sequences of the CD3γ/δ and CD3ɛ homologues in the mandarin fish, Siniperca chuats i, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ɛ contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ɛ showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N -glycosylation site was present in CD3ɛ, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ɛ subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ɛ in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ɛ were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ɛ were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ɛ mRNA, indicating that CD3γ/δ and CD3ɛ lymphocytes are involved in the immune response of this species.

  14. Altered structural connectivity of cortico-striato-pallido-thalamic networks in Gilles de la Tourette syndrome

    PubMed Central

    Marrakchi-Kacem, Linda; Lecomte, Sophie; Valabregue, Romain; Poupon, Fabrice; Guevara, Pamela; Tucholka, Alan; Mangin, Jean-François; Vidailhet, Marie; Lehericy, Stephane; Hartmann, Andreas; Poupon, Cyril

    2015-01-01

    Gilles de la Tourette syndrome is a childhood-onset syndrome characterized by the presence and persistence of motor and vocal tics. A dysfunction of cortico-striato-pallido-thalamo-cortical networks in this syndrome has been supported by convergent data from neuro-pathological, electrophysiological as well as structural and functional neuroimaging studies. Here, we addressed the question of structural integration of cortico-striato-pallido-thalamo-cortical networks in Gilles de la Tourette syndrome. We specifically tested the hypothesis that deviant brain development in Gilles de la Tourette syndrome could affect structural connectivity within the input and output basal ganglia structures and thalamus. To this aim, we acquired data on 49 adult patients and 28 gender and age-matched control subjects on a 3 T magnetic resonance imaging scanner. We used and further implemented streamline probabilistic tractography algorithms that allowed us to quantify the structural integration of cortico-striato-pallido-thalamo-cortical networks. To further investigate the microstructure of white matter in patients with Gilles de la Tourette syndrome, we also evaluated fractional anisotropy and radial diffusivity in these pathways, which are both sensitive to axonal package and to myelin ensheathment. In patients with Gilles de la Tourette syndrome compared to control subjects, we found white matter abnormalities in neuronal pathways connecting the cerebral cortex, the basal ganglia and the thalamus. Specifically, striatum and thalamus had abnormally enhanced structural connectivity with primary motor and sensory cortices, as well as paracentral lobule, supplementary motor area and parietal cortices. This enhanced connectivity of motor cortex positively correlated with severity of tics measured by the Yale Global Tics Severity Scale and was not influenced by current medication status, age or gender of patients. Independently of the severity of tics, lateral and medial orbito

  15. Altered structural connectivity of cortico-striato-pallido-thalamic networks in Gilles de la Tourette syndrome.

    PubMed

    Worbe, Yulia; Marrakchi-Kacem, Linda; Lecomte, Sophie; Valabregue, Romain; Poupon, Fabrice; Guevara, Pamela; Tucholka, Alan; Mangin, Jean-François; Vidailhet, Marie; Lehericy, Stephane; Hartmann, Andreas; Poupon, Cyril

    2015-02-01

    Gilles de la Tourette syndrome is a childhood-onset syndrome characterized by the presence and persistence of motor and vocal tics. A dysfunction of cortico-striato-pallido-thalamo-cortical networks in this syndrome has been supported by convergent data from neuro-pathological, electrophysiological as well as structural and functional neuroimaging studies. Here, we addressed the question of structural integration of cortico-striato-pallido-thalamo-cortical networks in Gilles de la Tourette syndrome. We specifically tested the hypothesis that deviant brain development in Gilles de la Tourette syndrome could affect structural connectivity within the input and output basal ganglia structures and thalamus. To this aim, we acquired data on 49 adult patients and 28 gender and age-matched control subjects on a 3 T magnetic resonance imaging scanner. We used and further implemented streamline probabilistic tractography algorithms that allowed us to quantify the structural integration of cortico-striato-pallido-thalamo-cortical networks. To further investigate the microstructure of white matter in patients with Gilles de la Tourette syndrome, we also evaluated fractional anisotropy and radial diffusivity in these pathways, which are both sensitive to axonal package and to myelin ensheathment. In patients with Gilles de la Tourette syndrome compared to control subjects, we found white matter abnormalities in neuronal pathways connecting the cerebral cortex, the basal ganglia and the thalamus. Specifically, striatum and thalamus had abnormally enhanced structural connectivity with primary motor and sensory cortices, as well as paracentral lobule, supplementary motor area and parietal cortices. This enhanced connectivity of motor cortex positively correlated with severity of tics measured by the Yale Global Tics Severity Scale and was not influenced by current medication status, age or gender of patients. Independently of the severity of tics, lateral and medial orbito

  16. Prolactin regulates transcription of the ion uptake Na+/Cl- cotransporter (ncc) gene in zebrafish gill

    USGS Publications Warehouse

    Breves, Jason P.; Serizier, Sandy B.; Goffin, Vincent; McCormick, Stephen D.; Karlstrom, Rolf O.

    2013-01-01

    Prolactin (PRL) is a well-known regulator of ion and water transport within osmoregulatory tissues across vertebrate species, yet how PRL acts on some of its target tissues remains poorly understood. Using zebrafish as a model, we show that ionocytes in the gill directly respond to systemic PRL to regulate mechanisms of ion uptake. Ion-poor conditions led to increases in the expression of PRL receptor (prlra), Na+/Cl− cotransporter (ncc; slc12a10.2), Na+/H+ exchanger (nhe3b; slc9a3.2), and epithelial Ca2+ channel (ecac; trpv6) transcripts within the gill. Intraperitoneal injection of ovine PRL (oPRL) increased ncc and prlra transcripts, but did not affect nhe3b or ecac. Consistent with direct PRL action in the gill, addition of oPRL to cultured gill filaments stimulated ncc in a concentration-dependent manner, an effect blocked by a pure human PRL receptor antagonist (Δ1-9-G129R-hPRL). These results suggest that PRL signaling through PRL receptors in the gill regulates the expression of ncc, thereby linking this pituitary hormone with an effector of Cl− uptake in zebrafish for the first time.

  17. Ammonia secretion from fish gill depends on a set of Rh glycoproteins.

    PubMed

    Nakada, Tsutomu; Westhoff, Connie M; Kato, Akira; Hirose, Shigehisa

    2007-04-01

    Ammonia excretion from the gill in teleost fish is essential for nitrogen elimination. Although numerous physiological studies have measured ammonia excretion, the mechanism of ammonia movement through the membranes of gill epithelial cells is still unknown. Mammalian Rh glycoproteins are members of a family of proteins that mediate ammonia transport in bacteria, yeast, and plants. We identified the Rh glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the pufferfish, Takifugu rubripes. Northern blot, in situ hybridization, and immunohistochemistry revealed that the pufferfish erythroid Rh glycoprotein homologue fRhag was present in red blood cells and the hematological organs (spleen and kidney) in fish. All four pufferfish Rh glycoproteins are specifically localized in the gill and line the pillar cells, pavement cells, and the mitochondrion-rich cells. Heterologous expression in Xenopus oocytes showed that they mediate methylammonium (an analog of ammonium) transport. These results suggest that pufferfish Rh glycoproteins are involved in ammonia excretion from the gill. These findings challenge the classic view that ammonia excretion in the fish gill occurs by passive diffusion.

  18. Tissue distributions of fluoride and its toxicity in the gills of a freshwater teleost, Cyprinus carpio.

    PubMed

    Cao, Jinling; Chen, Jianjie; Wang, Jundong; Wu, Xiangtian; Li, Yundong; Xie, Lingtian

    2013-04-15

    Fish take up fluoride directly from water and are susceptible to fluoride contamination of their environment. In this study, we examined the tissue distributions of fluoride and its toxicity in the gills of the common carp (Cyprinus carpio) chronically exposed to fluoride. Carp were exposed to a range of aqueous fluoride (35-124 mg/L) and sampled at 30, 60 and 90 days. The accumulation of fluoride in the tissues increased with the level and duration of exposure. Steady state was not achieved under the experimental conditions. The gills accumulated the highest levels of fluoride followed by the liver>brain>kidney>muscle>intestine. A dose-dependent inhibition was observed for the enzyme activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase in the gills after the fish were exposed for 90 days. Also, accumulation of fluoride was associated with the inhibition of superoxide dismutase (SOD) activities and a dose-dependent stimulation of malondialdehyde (MDA) levels in the gill tissues, suggesting that fluoride promoted oxidative stress in the fish. Microscopic examinations revealed injuries to gill tissues and chloride cells, with the severity of injury increasing with exposure concentration. These results suggest that chronic exposure to elevated concentrations of fluoride may induce toxicity in the common carp.

  19. New insights into the many functions of carbonic anhydrase in fish gills.

    PubMed

    Gilmour, Kathleen M

    2012-12-01

    Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible reactions of carbon dioxide and water: CO(2) + H(2)O ↔ H(+) + HCO(3)(-). It has long been recognized that CA is abundant in the fish gill, with attention focused on the role of CA in catalyzing the hydration of CO(2) to provide H(+) and HCO(3)(-) for the branchial ion transport processes that underlie systemic ionic and acid-base regulation. Recent work has explored the diversity of CA isoforms in the fish gill. By linking these isoforms to different cell types in the gill, and by exploiting the diversity of fish species available for study, this work is increasing our understanding of the many roles that CA plays in the fish gill. In particular, recent work has revealed that fish utilize more than one model of CO(2) excretion, that to understand the role of CA and the gill in ionic regulation and acid-base balance means characterizing the transporter and CA complement of individual cell types, and that CA plays roles in branchial sensory mechanisms. The goal of this brief review is to summarize these new developments, while at the same time highlighting key areas in which further research is needed.

  20. Hagfish: Champions of CO2 tolerance question the origins of vertebrate gill function

    PubMed Central

    Baker, Daniel W.; Sardella, Brian; Rummer, Jodie L.; Sackville, Michael; Brauner, Colin J.

    2015-01-01

    The gill is widely accepted to have played a key role in the adaptive radiation of early vertebrates by supplanting the skin as the dominant site of gas exchange. However, in the most basal extant craniates, the hagfishes, gills play only a minor role in gas exchange. In contrast, we found hagfish gills to be associated with a tremendous capacity for acid-base regulation. Indeed, Pacific hagfish exposed acutely to severe sustained hypercarbia tolerated among the most severe blood acidoses ever reported (1.2 pH unit reduction) and subsequently exhibited the greatest degree of acid-base compensation ever observed in an aquatic chordate. This was accomplished through an unprecedented increase in plasma [HCO3−] (>75 mM) in exchange for [Cl−]. We thus propose that the first physiological function of the ancestral gill was acid-base regulation, and that the gill was later co-opted for its central role in gas exchange in more derived aquatic vertebrates. PMID:26057989

  1. A Novel Betaproteobacterial Agent of Gill Epitheliocystis in Seawater Farmed Atlantic Salmon (Salmo salar)

    PubMed Central

    Toenshoff, Elena R.; Kvellestad, Agnar; Mitchell, Susan O.; Steinum, Terje; Falk, Knut; Colquhoun, Duncan J.; Horn, Matthias

    2012-01-01

    Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although ‘Candidatus Piscichlamydia salmonis’, previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as ‘Candidatus Branchiomonas cysticola’, was the cyst-forming agent in these samples. While histology and ultrastructure of ‘Ca. B. cysticola’ cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae. PMID:22427865

  2. Functional morphology of the gill in amazonian freshwater stingrays (chondrichthyes: potamotrygonidae): implications for adaptation to freshwater.

    PubMed

    Duncan, Wallice Paxiúba; da Costa, Oscar Tadeu Ferreira; Sakuragui, Marise Margareth; Fernandes, Marisa Narciso

    2010-01-01

    The gill morphologies of six species of potamotrygonid freshwater stingrays from the Amazon basin were investigated using light and electron microscopy. Some unique features were found in the potamotrygonid gill: (1) fingerlike protuberances on the gill filament, (2) an Alcian blue/periodic acid-Schiff-positive histochemical reaction for several cell layers in the gill epithelium (except the basal ones), (3) pavement cells with numerous subapical mucous vesicles, (4) very large mucous cells, and (5) follicular Na(+)/K(+)-ATPase-rich (NKA-rich) mitochondria-rich cells (MRCs) in Potamotrygon sp. (known as the cururu ray). The fingerlike protuberances may constitute an additional resistance to water flow, helping to drive water through the lamellae. The secretion of a mucous substance by the pavement cells and mucous cells may help to protect the gills against mechanical injury and pathogens and aid in osmoregulation in the dilute water of the Amazon basin. All MRCs possess enfolded basolateral membranes and have poorly developed or absent tubular systems. NKA-rich MRCs are located high in the basolateral membrane. The cururu ray, which is endemic to the Rio Negro, has follicular NKA-rich MRCs (8-12 cells in cross section) that share the same apical pit in the filament; this may be considered to be an autapomorphy. The combination of these branchial characteristics may have favored tolerance to the freshwater environment during the evolution and diversification of potamotrygonids throughout the Amazon basin.

  3. A structure-function analysis of ion transport in crustacean gills and excretory organs.

    PubMed

    Freire, Carolina A; Onken, Horst; McNamara, John C

    2008-11-01

    Osmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills, together with the excretory organs. In addition to their role in gas exchange, the gills constitute organs of active, transepithelial, ion transport, an activity of major importance that underlies many essential physiological functions like osmoregulation, calcium homeostasis, ammonium excretion and extracellular pH regulation. This review focuses on structure-function relationships in crustacean gills and excretory effectors, from the organ to molecular levels of organization. We address the diversity of structural architectures encountered in different crustacean gill types, and in constituent cell types, before examining the physiological mechanisms of Na(+), Cl(-), Ca(2+) and NH(4)(+) transport, and of acid-base equivalents, based on findings obtained over the last two decades employing advanced techniques. The antennal and maxillary glands constitute the principal crustacean excretory organs, which have received less attention in functional studies. We examine the diversity present in antennal and maxillary gland architecture, highlighting the structural similarities between both organ types, and we analyze the functions ascribed to each glandular segment. Emphasis is given to volume and osmoregulatory functions, capacity to produce dilute urine in freshwater crustaceans, and the effect of acclimation salinity on urine volume and composition. The microanatomy and diversity of function ascribed to gills and excretory organs are appraised from an evolutionary perspective, and suggestions made as to future avenues of investigation that may elucidate evolutionary and adaptive trends underpinning the invasion and exploitation of novel habitats.

  4. Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls.

    PubMed

    Duperron, Sébastien; Laurent, Mélina C Z; Gaill, Françoise; Gros, Olivier

    2008-03-01

    Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.

  5. Effects of gill-net trauma, barotrauma, and deep release on postrelease mortality of Lake Trout

    USGS Publications Warehouse

    Ng, Elizabeth L.; Fredericks, Jim P.; Quist, Michael C.

    2015-01-01

    Unaccounted postrelease mortality violates assumptions of many fisheries studies, thereby biasing parameter estimates and reducing efficiency. We evaluated effects of gill-net trauma, barotrauma, and deep-release treatment on postrelease mortality of lake trout Salvelinus namaycush. Lake trout were captured at depths up to 65 m with gill nets in Priest Lake, Idaho, and held in a large enclosure for 10–12 d. Postrelease mortality was the same for surface-release–and deep-release–treated fish (41%). Mixed-effects logistic regression models were used to evaluate effects of intrinsic and environmental factors on the probability of mortality. Presence of gill-net trauma and degree of barotrauma were associated with increased probability of postrelease mortality. Smaller fish were also more likely to suffer postrelease mortality. On average, deep-release treatment did not reduce postrelease mortality, but effectiveness of treatment increased with fish length. Of the environmental factors evaluated, only elapsed time between lifting the first and last anchors of a gill-net gang (i.e., lift time) was significantly related to postrelease mortality. Longer lift times, which may allow ascending lake trout to acclimate to depressurization, were associated with lower postrelease mortality rates. Our study suggests that postrelease mortality may be higher than previously assumed for lake trout because mortality continues after 48 h. In future studies, postrelease mortality could be reduced by increasing gill-net lift times and increasing mesh size used to increase length of fish captured.

  6. Relationship between biomass, seed components and seed Cd concentration in various peanut (Arachis hypogaea L.) cultivars grown on Cd-contaminated soils.

    PubMed

    Shi, Gangrong; Su, Gengqiang; Lu, Ziwei; Liu, Caifeng; Wang, Xvming

    2014-12-01

    Peanuts (Arachis hypogaea L.) exhibit high genotypic variations in seed Cd accumulation, but the mechanism remains unclear. This study aimed to reveal the main factors that determine Cd concentration in peanut seeds. The biomasses and Cd accumulation in plant tissues as well as the Cd distribution in the seeds of 15 peanut cultivars were analyzed in a pot experiment at 4mgkg(-1) Cd (treatment) and 0mgkg(-1) Cd (control). Peanuts exhibited large variations among cultivars in terms of Cd accumulation and distribution at the whole-plant and seed levels. The peanut cultivars were divided into three groups based on [Cd]embryos as follows: (i) high Cd accumulators (Zhenghong 3 and Haihua 1), (ii) low Cd accumulators (Qishan 208, Luhua 8, and Yuhua 15), and (iii) intermediate Cd accumulators (10 remaining cultivars). [Cd]embryos was significantly correlated with [Cd]testae and [Cd]oils at control conditions, whereas in the 4mgkg(-1) Cd treatment, [Cd]embryos was negatively correlated with plant biomass, total Cd and its proportion in vegetative organs, and seed oil contents. [Cd]embryos was positively correlated with protein contents, [Cd]oils, and proportion of Cd in protein extracts at 4mgkg(-1) Cd treatments. The attenuation of Cd by high biomass of vegetative tissues and Cd-binding proteins in seeds mainly determined the Cd concentration in peanut seeds.

  7. Ultrastructural observations on feeding appendages and gills of Alvinella pompejana (Annelida, Polychaeta)

    NASA Astrophysics Data System (ADS)

    Storch, V.; Gaill, F.

    1986-09-01

    The feeding appendages of Alvinella pompejana obtained from a deep-sea hydrothermal vent environment are described. They are characterized by a ciliated groove, the cells of which have a very distinctive ultrastructure, by groups of bipolar receptor cells and by several kinds of gland cells. Among these, one cell type is in an upside down position suggesting a function completely different from other epidermal secretory cells. The gills differ considerably from the feeding appendages on the basis of their ultrastructure. Their epidermis is very irregular in height; basal infoldings give the blood access to a space coming very near to the external medium. The blood vascular system is open. On the other hand, the gills of Amphicteis gunneri are not effective sites of gas exchange, since their columnar epithelium is underlain with muscle cells. The cells composing the feeding appendages and gills of Alvinella pompejana are characterized by ultrastructurally very different mitochondria.

  8. Non-electrolyte permeability of trout gills: effect of temperature and adrenaline

    PubMed Central

    Isaia, J.

    1979-01-01

    1. The gill permeability to various non-electrolytes (Ps) was measured in fresh-water and sea-water adapted trout (Salmo gairdneri). This study was performed in vitro using a `head-perfused' preparation. The influence of temperature and adrenaline (10-6 M) on permeability to non-electrolytes was also investigated. 2. During salt adaptation Pbutanol and Pwater decrease, Pmannitol rises and Pdextran stays constant. In view of recently acquired morphological data these results back up the hypothesis of different pathways across the gill epithelium (transcellular, vesicular and paracellular) according to the physico-chemical characteristics of the molecules. The low selectivity of the gill epithelium as a function of the liposolubility of the molecules used testifies to the hydrophilic nature of diffusion across this epithelium, a feature becoming more pronounced during salt adaptation. 3. The activation energies are about 4 kcal/mol, an energy comparable to diffusion in water for most of the substances tested, exceptions being butanol for fresh-water adapted gills and water for fresh-water and sea-water adapted gills. Arrhenius plots for butanol in fresh water gills show a transition temperature at 15 °C, suggesting an increased membrane lipid fluidity above this temperature. 4. Adrenaline has no effect on Pmannitol and Pdextran, but increases Pbutanol and Pwater selectively according to the adaptation medium (+ 160% and + 100% in fresh water and + 25% and + 20% in sea water respectively). These results point to an effect of this catecholamine on the membrane lipid fluidity. PMID:439031

  9. Cadmium resistance in Drosophila: a small cadmium binding substance

    SciTech Connect

    Jacobson, K.B.; Williams, M.W.; Richter, L.J.; Holt, S.E.; Hook, G.J.; Knoop, S.M.; Sloop, F.V.; Faust, J.B.

    1985-01-01

    A small cadmium-binding substance (CdBS) has been observed in adult Drosophila melanogaster that were raised for their entire growth cycle on a diet that contained 0.15 mM CdCl/sub 2/. Induction of CdBS was observed in strains that differed widely in their sensitivity of CdCl/sub 2/. This report describes the induction of CdBS and some of its characteristics. 17 refs., 4 figs., 1 tab.

  10. TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes.

    PubMed

    Salzmann, Steffen; Lang, Isabell; Rosenthal, Alevtina; Schäfer, Viktoria; Weisenberger, Daniela; Carmona Arana, José Antonio; Trebing, Johannes; Siegmund, Daniela; Neumann, Manfred; Wajant, Harald

    2013-09-01

    We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

  11. Forced Detachment of the CD2-CD58 Complex

    PubMed Central

    Bayas, M. V.; Schulten, K.; Leckband, D.

    2003-01-01

    The force-induced detachment of the adhesion protein complex CD2-CD58 was studied by steered molecular dynamics simulations. The forced detachment of CD2 and CD58 shows that the system can respond to an external force by two mechanisms, which depend on the loading rate. At the rapid loading rates of 70 and 35 pN/ps (pulling speeds of 1 and 0.5 Å/ps) the two proteins unfold before they separate, whereas at slower loading rates of 7 and 3.5 pN/ps (pulling speeds of 0.1 and 0.05 Å/ps), the proteins separate before the domains can unfold. When subjected to a constant force of 400 pN, the two proteins separated without significant structural distortion. These findings suggest that protein unfolding is not coupled to the adhesive function of CD2 and CD58. The simulations further confirm that salt bridges primarily determine the tensile strength of the protein-to-protein bond, and that the order of salt bridge rupture depends mainly on the position of the bond, relative to the line of action of the applied force. Salt bridges close to this line break first. The importance of each of the salt bridges for adhesion, determined from the simulations, correlates closely with their role in cell-to-cell adhesion and equilibrium binding determined by site-directed mutagenesis experiments. PMID:12668431

  12. Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

    PubMed Central

    Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

    2013-01-01

    The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

  13. Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary.

    PubMed

    Luo, Lianzhong; Ke, Caihuan; Guo, Xiaoyu; Shi, Bo; Huang, Miaoqin

    2014-06-01

    Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it

  14. Influence of cadmium concentration and length of exposure on metabolic rate and gill Na+/K+ ATPase activity of golden shiners (Notemigonus crysoleucas).

    PubMed

    Peles, John D; Pistole, David H; Moffe, Mickey

    2012-06-01

    Although metabolic rate is considered to be useful as a general indicator of the biological effects of exposure to metals, it is seldom measured in conjunction with specific physiological, biochemical or cellular parameters. The purpose of this investigation was to examine the influence of cadmium (Cd) exposure on metabolic rate and gill Na(+)/K(+) ATPase activity in golden shiners (Notemigonus crysoleucas). Shiners were exposed to six levels of Cd (ranging from control to the maximum sublethal concentration) for 24- and 96-h periods. After 24-h, metabolic rate and Na(+)/K(+) ATPase activity of individual fish were strongly correlated. Shiners exposed to the four highest Cd concentrations (500, 800, 1100, and 1400 μg L(-1)) for 24-h exhibited a shock response that was characterized by mean values for metabolic rate and Na(+)/K(+) ATPase activity that were significantly lower compared to the control. Although results for 96-h exposures reflect a repair/recovery phase, there was no significant correlation between metabolic rate and Na(+)/K(+) ATPase activity. Metabolic rate of shiners was significantly elevated (65-100%) at all concentrations compared to the control after 96-h, whereas Na(+)/K(+) ATPase activity did not differ from the control. Elevated metabolic rate after 96-h likely reflects the influence of a variety of energetically demanding processes associated with repair and recovery.

  15. Effects of salinity on the accumulation of hemocyte aggregates and bacteria in the gills of Callinectes sapidus, the Atlantic blue crab, injected with Vibrio campbellii.

    PubMed

    Ikerd, Jennifer L; Burnett, Karen G; Burnett, Louis E

    2015-05-01

    In addition to respiration and ion regulation, crustacean gills accumulate and eliminate injected particles, along with hemocyte aggregates that form in response to those particles. Here we report that the dose of Vibrio campbellii previously shown to induce a decrease in respiration and hemolymph flow across the gill in the Atlantic blue crab, Callinectes sapidus, also triggered the formation of aggregates containing four or more hemocytes in the gills, compared with saline-injected controls. More bacteria were trapped and rendered non-culturable per unit weight by anterior respiratory gills than posterior gills specialized for ion regulation. Further, more bacteria accumulated in the anterior gills of animals held at 30 ppt than those at 10 ppt. Thus, the role of the gills in immune defense comes at an energetic cost to this and likely to other crustaceans; this cost is influenced by acclimation salinity and the position and specialized function of individual gills.

  16. Structural Changes in the Somatosensory System Correlate with Tic Severity in Gilles de la Tourette Syndrome

    ERIC Educational Resources Information Center

    Thomalla, Gotz; Siebner, Hartwig R.; Jonas, Melanie; Baumer, Tobias; Biermann-Ruben, Katja; Hummel, Friedhelm; Gerloff, Christian; Muller-Vahl, Kirsten; Schnitzler, Alfons; Orth, Michael; Munchau, Alexander

    2009-01-01

    Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder characterized by multiple motor and vocal tics. Previous structural MRI studies have identified regional abnormalities in grey matter, especially in the basal ganglia. These findings are consistent with the assumption of alterations in cortico-striato-thalamo-cortical circuits and…

  17. Electronic Thesis Initiative: Pilot Project of McGill University, Montreal

    ERIC Educational Resources Information Center

    Park, Eun G.; Zou, Qing; McKnight, David

    2007-01-01

    Purpose: To set up a protocol for electronic thesis and dissertation (ETD) submission for the electronic thesis initiative pilot project at McGill University in Montreal, Canada. Design/methodology/approach: An electronic thesis and dissertation submission protocol was implemented and tested. To test authoring tools, we had 50 students submit…

  18. RESPIRATORY-CARDIOVASCULAR PHYSIOLOGY AND XENOBIOTIC GILL FLUX IN THE LAKE TROUT (SALVELINUS NAMAYCUSH)

    EPA Science Inventory

    An in vivo respirometer-metabolism chamber was used to obtain respiratory-cardiovascular physiology under normoxic and hypoxic conditions, and xenobiotic gill absorption (flux) data on adult lake trout (Salvelinus namaycush) over a 48-h exposure period at 11? 1?C.

  19. Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms.

    PubMed

    Hwang, Pung-Pung; Lee, Tsung-Han; Lin, Li-Yih

    2011-07-01

    Fish encounter harsh ionic/osmotic gradients on their aquatic environments, and the mechanisms through which they maintain internal homeostasis are more challenging compared with those of terrestrial vertebrates. Gills are one of the major organs conducting the internal ionic and acid-base regulation, with specialized ionocytes as the major cells carrying out active transport of ions. Exploring the iono/osmoregulatory mechanisms in fish gills, extensive literature proposed several models, with many conflicting or unsolved issues. Recent studies emerged, shedding light on these issues with new opened windows on other aspects, on account of available advanced molecular/cellular physiological approaches and animal models. Respective types of ionocytes and ion transporters, and the relevant regulators for the mechanisms of NaCl secretion, Na(+) uptake/acid secretion/NH(4)(+) excretion, Ca(2+) uptake, and Cl(-) uptake/base secretion, were identified and functionally characterized. These new ideas broadened our understanding of the molecular/cellular mechanisms behind the functional modification/regulation of fish gill ion transport during acute and long-term acclimation to environmental challenges. Moreover, a model for the systematic and local carbohydrate energy supply to gill ionocytes during these acclimation processes was also proposed. These provide powerful platforms to precisely study transport pathways and functional regulation of specific ions, transporters, and ionocytes; however, very few model species were established so far, whereas more efforts are needed in other species.

  20. Theohanellus toyamai infecting the gills of Koi cyprinus carpio in the Eastern United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A myxozoan resembling species of Thelohanellus was isolated from the gills of koi (Cyprinus carpio) cultured in North Carolina. Plasmodia measuring ~ 200µm in diameter contained tear-shaped myxospores containing a single pyriform polar capsule. The spore body was concave on one side, measuring 16....

  1. Thelohanellus toyamai infecting the gills of koi Cyprinus carpio in the eastern United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A myxozoan resembling species of Thelohanellus was isolated from the gills of koi (Cyprinus carpio) cultured in North Carolina. Plasmodia measuring ~ 200µm in diameter contained tear-shaped myxospores containing a singly pyriform polar capsule. The spore body was concave on one side, measuring, 1...

  2. Aquatic gilled mushrooms: Psathyrella fruiting in the Rogue River in southern Oregon.

    PubMed

    Frank, Jonathan L; Coffan, Robert A; Southworth, Darlene

    2010-01-01

    A species of Psathyrella (Basidiomycota) with true gills has been observed fruiting underwater in the clear, cold, flowing waters of the upper Rogue River in Oregon. Fruiting bodies develop and mature in the main channel, where they are constantly submerged, and were observed fruiting over 11 wk. These mushrooms develop underwater, not on wood recently washed into the river. Substrates include water-logged wood, gravel and the silty riverbed. DNA sequences of the ITS region and a portion of the ribosomal large subunit gene place this fungus in Psathyrella sensu stricto near P. atomata, P. fontinalis and P. superiorensis. Morphological characters distinguish the underwater mushroom from previously described species. Fruiting bodies have long fibrillose stipes with small diameter caps. Immature stages have a thin veil that is soon lost. Gills lack reddish edges. Cystidia are ventricose with subacute apices. Spores were observed as wedge-shape rafts released into gas pockets below the caps. Underwater gills and ballistospores indicate a recent adaptation to the stream environment. This particular river habitat combines the characteristics of spring-fed flows and cold, aerated water with woody debris in shallow depths on a fine volcanic substrate. Based on molecular and morphological evidence we conclude that the underwater mushrooms are a new species, Psathyrella aquatica. This report adds to the biodiversity of stream fungi that degrade woody substrates. The underwater environment is a new habitat for gilled mushrooms.

  3. Nonlethal gill biopsy does not affect juvenile chinook salmon implanted with radio transmitters

    USGS Publications Warehouse

    Martinelli-Liedtke, T. L.; Shively, R.S.; Holmberg, G.S.; Sheer, M.B.; Schrock, R.M.

    1999-01-01

    Using gastric and surgical transmitter implantation, we compared radio-tagged juvenile chinook salmon Oncorhynchus tshawytscha (T(O)) with tagged fish also having a gill biopsy (T(B)) to determine biopsy effects on fish implanted with radio transmitters. We found no evidence during the 21-d period to suggest that a gill biopsy reduced survival, growth, or gross condition of the tagged-biopsy group, regardless of transmitter implantation technique. We recorded 100% survival of all treatment groups. Relative growth rates of T(O) and T(B) fish did not differ significantly. Leukocrit and lysozyme levels were not significantly different among groups, suggesting that no signs of infection were present. Our findings suggest that small chinook salmon can tolerate the combination of transmitter implantation and gill biopsy without compromising condition relative to fish receiving only the transmitter. We believe a gill biopsy can be used in field telemetry studies, especially when physiological data are needed in addition to behavioral data.

  4. The Problematics of Human Subjectivity: Gilles Deleuze and the Deweyan Legacy.

    ERIC Educational Resources Information Center

    Semetsky, Inna

    2003-01-01

    Explores continuity between the philosophical positions: French poststructuralist Gilles Deleuze and John Dewey. Aims to open up consideration of Deleuze's philosophy to educational theory and practice in the context of current debates and in line with Dewey's legacy. Concludes the paper by affirming Deleuze's place in the contemporary scholarship…

  5. Increased daylength stimulates plasma growth hormone and gill Na+, K+ and -ATPase Atlantic salmon (Salmo salar )

    USGS Publications Warehouse

    McCormick, S.D.; Bjornsson, Bjorn Thrandur; Sheridan, M.; Eilertson, C.; Carey, J.B.; O'Dea, M.

    1995-01-01

    Atlantic salmon juveniles reared at constant temperature (9–10°C) were exposed to four photoperiod treatment and sampled every 2 weeks from January through May. Fish reared under normal photoperiod exhibited eight-and three fold increases in plasma growth hormone and gill Na+, K+-ATPase activity, respectively, between January and April. Fish exposed to abrupt increases in daylength (LD 15:9) in February or March responded with earlier increases in plasma growth hormone and gill Na+, K+-ATPase activity, and earlier decreases in condition factor relative to fish in the normal photoperiod group. Fish maintained under short daylength (LD 9:15) from January to May exhibited delayed and muted increases in plasma growth hormone and gill Na+, K+-ATPase activity. Plasma thyroxine exhibited a 2.5-fold increase from February to late March in the normal photoperiod group, was generally lower in the LD 9:15 group, but exhibited no obvious response to abrupt increases in daylength. There was an increase in plasma 3,5,3′-triiodo-l-thyronine with time in all groups (43–80%) but no significant response to photoperiod. Plasma levels of somatostatin-25 were highest in the LD 9:15 group, but there was no detectable response to increased daylength in any of the photoperiod treatments. The results indicate that plasma growth hormone is responsive to increased daylength and may be causally related to subsequent increases in gill Na+, K+-ATPase.

  6. Climatic variability in combination with eutrophication drives adaptive responses in the gills of Lake Victoria cichlids.

    PubMed

    van Rijssel, Jacco C; Hecky, Robert E; Kishe-Machumu, Mary A; Meijer, Saskia E; Pols, Johan; van Tienderen, Kaj M; Ververs, Jan D; Wanink, Jan H; Witte, Frans

    2016-12-01

    Textbook examples of adaptive radiation often show rapid morphological changes in response to environmental perturbations. East Africa's Lake Victoria, famous for its stunning adaptive radiation of cichlids, has suffered from human-induced eutrophication over the past decades. This cultural eutrophication is thought to be partly responsible for the dramatically reduced cichlid biodiversity, but climatic variability in itself might also have contributed to the eutrophication which resulted in low oxygen levels and decreased water transparency. To determine how recent environmental changes have influenced the lake and its cichlids over the past 50 years, we gathered environmental and meteorological variables and compared these with gill surface area of four cichlid species. We found that during the period of severe eutrophication and temperature increase (1980s), reduced wind speeds coincided with a reduction in oxygen levels and a decrease in both water temperature and transparency. The gill surface area in three out of the four cichlid species increased during this period which is consistent with adaptive change in response to increased hypoxia. During the 2000s, wind speeds, oxygen levels, water transparency and water temperature increased again, while cichlid gill surface area decreased. Our results imply that climatic changes and especially wind speed and direction might play a crucial role in tropical lake dynamics. The changes in Lake Victoria's water quality coincide with fluctuations in cichlid gill surface area, suggesting that these fish can respond rapidly to environmental perturbations, but also that climatic variability, together with continued eutrophication, might be detrimental to the lake's cichlid biodiversity.

  7. Isolation and characterization of bioactive fungi from shark Carcharodon carcharias' gill with biopharmaceutical prospects

    NASA Astrophysics Data System (ADS)

    Zhang, Yi; Han, Jinyuan; Feng, Yan; Mu, Jun; Bao, Haiyan; Kulik, Andreas; Grond, Stephanie

    2016-01-01

    Until recently, little was known about the fungi found in shark gills and their biomedicinal potential. In this article, we described the isolation, bioactivity, diversity, and secondary metabolites of bioactive fungi from the gill of a shark ( Carcharodon carcharias). A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated significant activity in four antimicrobial, pesticidal, and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit (ITS-LSU) rDNA-sequence BLAST comparison. Four genera, Penicillium, Aspergillus, Mucor, and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profiles. These results detail the broad range of bioactive fungi found in a shark's gills, revealing their biopharmaceutical potential. To the best of our knowledge, this is the first study characterizing shark gill fungi and their bioactivity.

  8. Gill remodelling and growth rate of striped catfish Pangasianodon hypophthalmus under impacts of hypoxia and temperature.

    PubMed

    Phuong, Le My; Huong, Do Thi Thanh; Nyengaard, Jens Randel; Bayley, Mark

    2017-01-01

    Gill morphometric and gill plasticity of the air-breathing striped catfish (Pangasianodon hypophthalmus) exposed to different temperatures (present day 27°C and future 33°C) and different air saturation levels (92% and 35%) during 6weeks were investigated using vertical sections to estimate the respiratory lamellae surface areas, harmonic mean barrier thicknesses, and gill component volumes. Gill respiratory surface area (SA) and harmonic mean water - blood barrier thicknesses (HM) of the fish were strongly affected by both environmental temperature and oxygen level. Thus initial values for 27°C normoxic fish (12.4±0.8g) were 211.8±21.6mm(2)g(-1) and 1.67±0.12μm for SA and HM respectively. After 5weeks in same conditions or in the combinations of 33°C and/or PO2 of 55mmHg, this initial surface area scaled allometrically with size for the 33°C hypoxic group, whereas branchial SA was almost eliminated in the 27°C normoxic group, with other groups intermediate. In addition, elevated temperature had an astounding effect on growth with the 33°C group growing nearly 8-fold faster than the 27°C fish.

  9. We Walked Very Warily. A History of Women at McGill.

    ERIC Educational Resources Information Center

    Gillett, Margaret

    Nineteenth-century assumptions about the nature of women, her educability, her role in society, and debates about coeducation are examined in this record of the efforts of the first women students to gain entrance in McGill University in Canada. The introduction offers historical background, ideas of women, and the opening of higher education to…

  10. Student Guide Bibliographies for the McLennan Library, McGill University.

    ERIC Educational Resources Information Center

    McGill Univ., Montreal (Quebec). McLennan Library.

    The McLennan Library of McGill University has created these student guide bibliographies as library orientation tools. They cover reference materials in specific subject areas in the social science and humanities, with special reference to their locations in McLennan Library. The guides are intended for the master's level student and have proved…

  11. Jacqueline Baxter Talks to Gill Howland, Newly Appointed Chair of BELMAS

    ERIC Educational Resources Information Center

    Baxter, Jacqueline

    2017-01-01

    Gill is currently Chair of the British Educational Leadership, Management and Administration Society. Her personal experiences are central to her belief that education is the key to unlocking potential, both for individuals and for society as a whole. Throughout her career she has championed the right to good quality, inspirational education for…

  12. Gills Onions Advanced Energy Recovery System: Turning a Waste Liability into a Renewable Resource

    DTIC Science & Technology

    2011-01-13

    Anaerobic Municipal Solid Waste Food Waste from Residential & Food Service Digestion Fats, Oil, and Grease...FOG) from Food Service Anaerobic Methane Wastewater Treatment Bi lid Digestion Fuel Cells oso s Think Holistically! Your Take Away Points...Gills Onions Advanced Energy Recovery System Turning a Waste Liability into a Renewable Resource Waste to Energy Using Fuel Cells

  13. Effects of dietary protein levels on the disease resistance, immune function and physical barrier function in the gill of grass carp (Ctenopharyngodon idella) after challenged with Flavobacterium columnare.

    PubMed

    Xu, Jing; Feng, Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2016-10-01

    The effects of dietary protein levels on the disease resistance, gill immune function and physical barrier function of grass carp (Ctenopharyngodon idella) were investigated in this study. A total of 540 grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that optimal levels of dietary protein had the following effects: (1) the production of antibacterial components increased, and anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels were up-regulated, whereas mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, IκB kinase (IKK) α, IKKβ, IKKγ, eIF4E-binding proteins (4E-BP) 1 and 4E-BP2 were down-regulated in the gills of grass carp (P < 0.05), indicating that fish gill immune function was enhanced at an optimal level of dietary protein; (2) the activities and mRNA levels of antioxidant enzymes and glutathione content increased, the contents of reactive oxygen species, malondialdehyde and protein carbonyl (PC) decreased, and NF-E2-related factor 2, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and tight junction complexes mRNA levels were up-regulated, whereas Kelch-like-ECH-associated protein (Keap) 1a, Keap1b, cysteinyl aspartic acid-protease 3, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, c-Jun N-terminal protein kinase, myosin light chain kinase and p38 mitogen-activated protein kinase mRNA levels were down-regulated in the gills of grass carp (P < 0.05), indicating that the fish gill physical barrier function improved at an optimal level of dietary protein. Finally, based on the gill rot morbidity, ACP activity and PC

  14. Synthesis and Spectral Studies of CdTe-Dendrimer Conjugates

    NASA Astrophysics Data System (ADS)

    Ghosh, Srabanti; Saha, Abhijit

    2009-08-01

    In order to couple high cellular uptake and target specificity of dendrimer molecule with excellent optical properties of semiconductor nanoparticles, the interaction of cysteine-capped CdTe quantum dots with dendrimer was investigated through spectroscopic techniques. NH2-terminated dendrimer molecule quenched the photoluminescence of CdTe quantum dots. The binding constants and binding capacity were calculated, and the nature of binding was found to be noncovalent. Significant decrease in luminescence intensity of CdTe quantum dots owing to noncovalent binding with dendrimer limits further utilization of these nanoassemblies. Hence, an attempt is made, for the first time, to synthesize stable, highly luminescent, covalently linked CdTe-Dendrimer conjugate in aqueous medium using glutaric dialdehyde (G) linker. Conjugate has been characterized through Fourier transform infrared spectroscopy and transmission electron microscopy. In this strategy, photoluminescence quantum efficiency of CdTe quantum dots with narrow emission bandwidths remained unaffected after formation of the conjugate.

  15. Identification of Methanotrophic Lipid Biomarkers in Cold-Seep Mussel Gills: Chemical and Isotopic Analysis

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Summons, Roger E.; Dowling, Lesley M.; Zahiralis, Karen D.

    1995-01-01

    A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta-8, delta-10, and delta-ll. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol(11.0% 4(alpha)-methyl-cholesta-8(14), 24-dien-3(beta)-ol) was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3(beta)-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4 per thousand for total tissue, -60.6 and -62.4 per thousand for total lipids, -60.2 and -63.9 per thousand for phospholipid fatty acids, and -71.8 and -73.8 per thousand for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria further supporting the conversion of the bacterial methyl-sterol pool.

  16. Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias).

    PubMed

    Gilmour, K M; Bayaa, M; Kenney, L; McNeill, B; Perry, S F

    2007-01-01

    Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.

  17. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system.

    PubMed

    Nebel, Catherine; Romestand, Bernard; Nègre-Sadargues, Geneviève; Grousset, Evelyse; Aujoulat, Fabien; Bacal, Julien; Bonhomme, François; Charmantier, Guy

    2005-10-01

    The effects of long-term freshwater acclimatization were investigated in juvenile sea-bass Dicentrarchus labrax to determine whether all sea-bass juveniles are able to live in freshwater and to investigate the physiological basis of a successful adaptation to freshwater. This study particularly focused on the ability of sea-bass to maintain their hydromineral balance in freshwater and on their ion (re)absorbing abilities through the gills and kidneys. Two different responses were recorded after a long-term freshwater acclimatization. (1) Successfully adapted sea-bass displayed standard behavior; their blood osmolality was maintained almost constant after the freshwater challenge, attesting to their efficient hyperosmoregulation. Their branchial and renal Na+/K+-ATPase abundance and activity were high compared to seawater fish due to a high number of branchial ionocytes and to the involvement of the urinary system in active ion reabsorption, producing hypotonic urine. (2) Sea-bass that had not successfully adapted to freshwater were recognized by abnormal schooling behavior. Their blood osmolality was low (30% lower than in the successfully adapted sea-bass), which is a sign of acute osmoregulatory failure. High branchial Na+/K+-ATPase abundance and activity compared to successfully adapted fish were coupled to a proliferation of gill chloride cells, whose ultrastructure did not display pathological signs. The large surface used by the gill chloride cells might negatively interfere with respiratory gas exchanges. In their urinary system, enzyme abundance and activity were low, in accordance with the observed lower density of the kidney tubules. Urine was isotonic to blood in unsuccessfully adapted fish, ruling out any participation of the kidney in hyperosmoregulation. The kidney failure seems to generate a compensatory ion absorption through increased gill activity, but net ion loss through urine seems higher than ion absorption by the gills, leading to lower hyper

  18. Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

    PubMed Central

    Yu, A. S. L.; Li, J.; Madsen, S. S.; Færgeman, N. J.

    2012-01-01

    Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na+-K+-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium. PMID:21975646

  19. Organization of the human CD9 gene

    SciTech Connect

    Rubinstein, E.; Benoit, P.; Billard, M.; Plaisance, S.; Prenant, M.; Boucheix, C. ); Uzan, G. )

    1993-04-01

    The CD9 antigen was originally described as a 24-kDa molecule present on B-lineage-derived acute lymphoblastic leukemia cells and developing B lymphocytes. Platelets also express a large amount of CD9 antigen and can be activated by CD9 antibodies. The authors report here the structure of the CD9 gene, which is composed of 8 exons spanning more than 20 kb. There is no TATA or CAAT box in the 5[prime]-flanking domain of the CD9 gene, but a 120-bp region extremely rich in C and G (88%) contains several Sp1 binding sites and a consensus site for the binding of zinc-finger proteins of the Krox/EGR family. The CD9 antigen belongs to a new cell surface protein family. The organization of its gene closely resembles the organization of the genes for two other members of this protein family, TAPA1 and CD63, which share with CD9 respectively 45 and 25% identity at the amino acid level. 55 refs., 5 figs.

  20. CD1 antigen presentation: how it works.

    PubMed

    Barral, Duarte C; Brenner, Michael B

    2007-12-01

    The classic concept of self-non-self discrimination by the immune system focused on the recognition of fragments from proteins presented by classical MHC molecules. However, the discovery of MHC-class-I-like CD1 antigen-presentation molecules now explains how the immune system also recognizes the abundant and diverse universe of lipid-containing antigens. The CD1 molecules bind and present amphipathic lipid antigens for recognition by T-cell receptors. Here, we outline the recent advances in our understanding of how the processes of CD1 assembly, trafficking, lipid-antigen binding and T-cell activation are achieved and the new insights into how lipid antigens differentially elicit CD1-restricted innate and adaptive T-cell responses.

  1. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. Results Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite

  2. Mitochondrion-rich cells distribution, Na+/K+-ATPase activity and gill morphometry of the Amazonian freshwater stingrays (Chondrichthyes: Potamotrygonidae).

    PubMed

    Duncan, Wallice P; Silva, Naara F; Fernandes, Marisa N

    2011-09-01

    Detailed measurements of gill area and constituent variables (total filament number, total filament length and mean filament length), and immunolocalization of the α-subunit of Na⁺/K⁺-ATPase and Na⁺/K⁺-ATPase activity were performed on both hemibranchs of all five arches of freshwater potamotrygonid stingrays (Paratrygon aiereba and Potamotrygon sp.). Both species exhibit similar mass-specific gill area, 89.8 ± 6.6 and 91.5 ± 4.3 mm² g⁻¹ for P. aiereba and Potamotrygon sp., respectively. The density of Na⁺/K⁺-ATPase-rich MRCs and Na⁺/K⁺-ATPase activity was higher in the 4th gill arch in both species. The Na⁺/K⁺-ATPase activity was positively correlated to the Na⁺/K⁺-ATPase-rich Na⁺/K⁺-ATPase rich) mitochondrion-rich cell (MRC) distribution among the gill arches of P. aiereba but not in Potamotrygon sp. The levels Na⁺/K⁺-ATPase activity were not correlated to the gill surface area among the arches for both rays' species. Considering that the Na⁺/K⁺-ATPase-rich MRC is the main site for active ion transport in the gill epithelia and Na⁺/K⁺-ATPase activity plays a crucial role in osmoionoregulatory function, we suggesting that 4th gill arch is more relevant for osmoregulation and ion balance in these potamotrygonids.

  3. EROD activity in gill filaments of anadromous and marine fish as a biomarker of dioxin-like pollutants.

    PubMed

    Jönsson, Maria; Abrahamson, Alexandra; Brunström, Björn; Brandt, Ingvar; Ingebrigtsen, Kristian; Jørgensen, Even H

    2003-11-01

    The applicability of a gill filament-based ethoxyresorufin O-deethylase (EROD) assay, originally developed in rainbow trout, was examined in Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens) and spotted wolffish (Anarhichas minor). All species but spotted wolffish showed strong EROD induction in tip pieces of gill filaments following 48 h of exposure to waterborne beta-naphthoflavone. Atlantic salmon parr, smolts held in freshwater and smolts transferred to seawater showed EROD induction of similar magnitude. Arctic charr, differing 11-fold in body weight, showed similar EROD activities as expressed per gill filament tip. Laboratory exposure of saithe to water and sediments collected at polluted sites, resulted in strong EROD induction. In conclusion, the gill filament assay seems useful for monitoring exposure to aryl hydrocarbon receptor agonists in various species. Furthermore, smoltification status, water salinity and body size proved to have minor influence on gill filament EROD activity. However, the results in spotted wolffish show that some species may be less suitable for monitoring using the gill assay. Assessment of gill filament EROD activity in fish exposed to polluted water and sediments in the laboratory proved to be an easy and cost-effective way to survey pollution with dioxin-like chemicals.

  4. Is gill cortisol concentration a good acute stress indicator in fish? A study in rainbow trout and zebrafish.

    PubMed

    Gesto, Manuel; Hernández, Juan; López-Patiño, Marcos A; Soengas, José L; Míguez, Jesús M

    2015-10-01

    Cortisol is the main biomarker of physiological stress in fish. It is usually measured in plasma, which requires blood collection. Though cortisol is produced in the anterior kidney, it can diffuse easily through cell membranes due to its lipophilic nature. Taking advantage of that, some non-invasive techniques have been developed to measure cortisol directly in the water from fish-holding tanks, in skin mucus or in scales. In this study, we explored the possibility to analyze fish cortisol from gill filaments as a reliable acute stress marker. Our results show that gill cortisol levels correlate well with plasma cortisol levels in both rainbow trout and zebrafish exposed or not to an acute stress protocol. Measuring cortisol in gill filaments increases the available possibilities for stress assessment in fish. Although this approach should yet be tested for its use with other stressors, it has several advantages: In relatively large fish (i.e. above 30 g) gill cortisol levels could be measured in vivo. Sampling of gill biopsies is very fast and easy, and the procedure does not induce stress if properly performed, making it an ideal option for in vivo stress assessment. In small fish, the use of gill tissue to measure cortisol has important technical advantages with respect to the current methods using whole-body homogenates. Gill homogenates could be used directly for ELISA cortisol analysis, avoiding the need of tedious and expensive cortisol extraction protocols, and, since no organic solvent is required, contributing for a more environmentally friendly analysis.

  5. Bacterial microbiota profile in gills of modified atmosphere-packaged oysters stored at 4 °C.

    PubMed

    Chen, Huibin; Wang, Meiying; Lin, Xiangzhi; Shi, Caihua; Liu, Zhiyu

    2017-02-01

    As filter-feeding bivalves, oysters can accumulate microorganisms into their gills, causing spoilage and potential safety issues. This study aims to investigate the changes in the gill microbiota of oysters packed under air and modified atmospheres (MAs, 50% CO2: 50% N2, 70% CO2: 30% O2, and 50% CO2: 50% O2) during storage at 4 °C. The diversity of bacterial microbiota in oyster gills was profiled through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis on the 16S rRNA gene V3 region to describe the variation during the entire storage period. The DGGE profile revealed high bacterial diversity in the air- and MA-packaged oyster gills, and the spoilage bacterial microbiota varied in the MA-packaged oyster gills. Results indicated that CO2:O2 (70%:30%) was suitable for oyster MA packaging and that high bacterial loads in oyster gills need to be considered during storage. In addition, Lactobacillus and Lactococcus species were found to grow dominantly in fresh oyster gills under MA packaging, which supports the potential application of MA packaging for oyster storage.

  6. Copper accumulation and toxicity in isolated cells from gills and hepatopancreas of the blue crab (Callinectes sapidus).

    PubMed

    Paganini, Christianne L; Bianchini, Adalto

    2009-06-01

    In the present study, we used fresh preparations of mixed-cell populations to evaluate accumulation and toxicity of dissolved copper (1-100 microM) in isolated cells from posterior gills and hepatopancreas of the blue crab (Callinectes sapidus). For both gill and hepatopancreatic cells, significant increases in copper accumulation were observed after exposure to 50 or 100 microM copper. In gill cells, a linear increase in copper accumulation was observed over time. In hepatopancreatic cells, a maximum level of copper accumulation was achieved after 1 h of exposure, remaining unchanged up to 6 h. After 6 h of exposure, copper content in gill cells was 6.6-fold higher than that in hepatopancreatic cells. In both cell types, copper accumulation always followed a linear relationship with copper concentration in the incubation medium. Significant decreases in cell viability were observed after exposure to either 10 microM copper (gill cells) or 100 microM copper (gill and hepatopancreatic cells). Furthermore, an exponential rise to maximum-type relationship was observed between copper accumulation and toxicity in gill cells. Altogether, these findings indicate that the premise behind the biotic ligand model (BLM) approach is verified in isolated cells from posterior gills of the blue crab (i.e., toxicity is driven by copper accumulation in the biotic ligand, the gill cell). Therefore, these cells can be used as a model for the development of an in vitro BLM version for marine conditions. Isolated cells from the hepatopancreas, however, could be used as a model to better understand the mechanism of copper tolerance at a cellular level in crustaceans.

  7. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium model alter in response to acute seawater exposure.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2015-11-01

    Gill epithelium permeability and qualitative/quantitative aspects of gill claudin (cldn) tight junction (TJ) protein transcriptomics were examined with a primary cultured model gill epithelium developed using euryhaline puffer fish (Tetraodon nigroviridis) gills. The model was prepared using seawater-acclimated fish gills and was cultured on permeable cell culture filter supports. The model is composed of 1-2 confluent layers of gill pavement cells (PVCs), with the outer layer exhibiting prominent apical surface microridges and TJs between adjacent cells. During development of electrophysiological characteristics, the model exhibits a sigmoidal increase in transpithelial resistance (TER) and plateaus around 30 kΩcm(2). At this point paracellular movement of [(3)H]polyethylene glycol (PEG) 4000 was low at ~1.75 cm s(-1)×10(-7). When exposed to apical seawater (SW) epithelia exhibit a marked decrease in TER while PEG flux remained unchanged for at least 6 h. In association with this, transcript encoding cldn TJ proteins cldn3c, -23b, -27a, -27c, -32a and -33b increased during the first 6 h while cldn11a decreased. This suggests that these proteins are involved in maintaining barrier properties between gill PVCs of SW fishes. Gill cldn mRNA abundance also altered 6 and 12 h following abrupt SW exposure of puffer fish, but in a manner that differed qualitatively and quantitatively from the cultured model. This most likely reflects the cellular heterogeneity of whole tissue and/or the contribution of the endocrine system in intact fish. The current study provides insight into the physiological and transcriptomic response of euryhaline fish gill cells to a hyperosmotic environment.

  8. Cd(2+) extrusion by P-type Cd(2+)-ATPase of Staphylococcus aureus 17810R via energy-dependent Cd(2+)/H(+) exchange mechanism.

    PubMed

    Tynecka, Zofia; Malm, Anna; Goś-Szcześniak, Zofia

    2016-08-01

    Cd(2+) is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd(2+)-resistant due to possession of cadA-coded Cd(2+) efflux system, recognized here as P-type Cd(2+)-ATPase. This Cd(2+) pump utilizing cellular energy-ATP, ∆μ H (+) (electrochemical proton potential) and respiratory protons, extrudes Cd(2+) from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd(2+) extrusion remains unknown. Here we propose that two Cd(2+) taken up by strain 17810R via Mn(2+) uniporter down membrane potential (∆ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high PiB) are trapped probably by high affinity sites in cytoplasmic domain of Cd(2+)-ATPase, forming SCdS. This stops Cd(2+) transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of Pi-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ∆ψ, extrude two trapped cytoplasmic Cd(2+), which move to low affinity sites, being then extruded into extracellular space via ∆ψ-dependent Cd(2+)/H(+) exchange. In 1 mM phosphate buffer (low PiB), external Cd(2+) competing with decreased number of Pi-dependent protons, binds to ψs of Cd(2+)-ATPase channel, enters cytoplasm through the channel down ∆ψ via Cd(2+)/Cd(2+) exchange and blocks dithiols in ODHC. However, Mg(2+) pretreatment preventing external Cd(2+) countertransport through the channel down ∆ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd(2+) via Cd(2+)/H(+) exchange, despite low PiB.

  9. The T cell antigen receptor CD3:CD4 molecular complex is diminished on the surface of pulmonary lymphocytes.

    PubMed Central

    Marathias, K.; Pinto, C.; Rodberg, G.; Preffer, F.; Wong, J.; Kradin, R.

    1994-01-01

    CD4, a 55-kd cell surface glycoprotein, binds to class II major histocompatibility complex (MHC) (Ia) antigens and functions as a coreceptor for the T cell antigen receptor (Ti alpha beta)-CD3 complex. We have observed that critical elements of the T cell antigen multireceptor complex, including Ti alpha beta, CD3, CD4, but not CD8, were diminished on CD45RO+ pulmonary T lymphocytes but not CD45RO+ peripheral blood T lymphocytes (PBL). Epitopes mapping from the first (D1) to the fourth (D4) extracytoplasmic Ig-like domains of CD4 were expressed to a lesser degree on pulmonary T cells than on PBL (P = 0.002). CD4 expression on pulmonary T cells did not increase after 72 hours of ex vivo culture in complete medium but was restored toward control levels by stimulation with phytohemagglutinin, anti-CD3, or interleukin-2. CD4 mRNA isolated from lung T cells and PBL co-migrated on Northern blots and the total levels of CD4 mRNA were comparable, suggesting that diminished CD4 expression by pulmonary T cells might reflect a posttranscriptional change. To determine whether CD4bright T cells convert with mitogen stimulation to CD4dim cells, PBLs were stimulated with immobilized anti-CD3, anti-CD4, or a molecularly engineered anti-CD3:CD4 bispecific monoclonal antibody and the ratio of the CD4:CD3 mean fluorescence staining intensities was calculated at days 3 and 13. The CD4:CD3 ratio decreased primarily for cells stimulated with anti-CD3:CD4, suggesting that co-ligation of CD3 and CD4 is required for the generation of CD4dim T cells. We conclude that diminished Ti alpha beta-CD3:CD4 expression is a characteristic of T cells in lung that is not shared by peripheral blood T cells in vivo, and speculate that this change reflects T cell activation in a millieu of limited interleukin-2 availability. Images Figure 8 Figure 9 PMID:7977652

  10. Gill transcriptome response to changes in environmental calcium in the green spotted puffer fish

    PubMed Central

    2010-01-01

    Background Calcium ion is tightly regulated in body fluids and for euryhaline fish, which are exposed to rapid changes in environmental [Ca2+], homeostasis is especially challenging. The gill is the main organ of active calcium uptake and therefore plays a crucial role in the maintenance of calcium ion homeostasis. To study the molecular basis of the short-term responses to changing calcium availability, the whole gill transcriptome obtained by Super Serial Analysis of Gene Expression (SuperSAGE) of the euryhaline teleost green spotted puffer fish, Tetraodon nigroviridis, exposed to water with altered [Ca2+] was analysed. Results Transfer of T. nigroviridis from 10 ppt water salinity containing 2.9 mM Ca2+ to high (10 mM Ca2+ ) and low (0.01 mM Ca2+) calcium water of similar salinity for 2-12 h resulted in 1,339 differentially expressed SuperSAGE tags (26-bp transcript identifiers) in gills. Of these 869 tags (65%) were mapped to T. nigroviridis cDNAs or genomic DNA and 497 (57%) were assigned to known proteins. Thirteen percent of the genes matched multiple tags indicating alternative RNA transcripts. The main enriched gene ontology groups belong to Ca2+ signaling/homeostasis but also muscle contraction, cytoskeleton, energy production/homeostasis and tissue remodeling. K-means clustering identified co-expressed transcripts with distinct patterns in response to water [Ca2+] and exposure time. Conclusions The generated transcript expression patterns provide a framework of novel water calcium-responsive genes in the gill during the initial response after transfer to different [Ca2+]. This molecular response entails initial perception of alterations, activation of signaling networks and effectors and suggests active remodeling of cytoskeletal proteins during the initial acclimation process. Genes related to energy production and energy homeostasis are also up-regulated, probably reflecting the increased energetic needs of the acclimation response. This study is the

  11. Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth bass (Micropterus salmoides)

    USGS Publications Warehouse

    Walsh, Heather L.; Iwanowicz, Luke R.; Glenney, Gavin W.; Iwanowicz, Deborah D.; Blazer, Vicki

    2012-01-01

    Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1–15.1) µm and width of 6.9 ± 1.1 (4.0–9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya, although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2–12.2) µm and width of 10.6 ± 0.6 (9.0–11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai. The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum, which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected

  12. Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass.

    PubMed

    Walsh, Heather L; Iwanowicz, Luke R; Glenney, Gavin W; Iwanowicz, Deborah D; Blazer, Vicki S

    2012-04-01

    Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a

  13. Multiple In Vitro and In Vivo Regulatory Effects of Budesonide in CD4+ T Lymphocyte Subpopulations of Allergic Asthmatics

    PubMed Central

    Pace, Elisabetta; Di Sano, Caterina; La Grutta, Stefania; Ferraro, Maria; Albeggiani, Giuseppe; Liotta, Giuseppe; Di Vincenzo, Serena; Uasuf, Carina Gabriela; Bousquet, Jean; Gjomarkaj, Mark

    2012-01-01

    Background Increased activation and increased survival of T lymphocytes characterise bronchial asthma. Objectives In this study the effect of budesonide on T cell survival, on inducible co-stimulator T cells (ICOS), on Foxp3 and on IL-10 molecules in T lymphocyte sub-populations was assessed. Methods Cell survival (by annexin V binding) and ICOS in total lymphocytes, in CD4+/CD25+ and in CD4+/CD25- and Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25-cells was evaluated, by cytofluorimetric analysis, in mild intermittent asthmatics (n = 19) and in controls (n = 15). Allergen induced T lymphocyte proliferation and the in vivo effects of budesonide in mild persistent asthmatics (n = 6) were also explored. Results Foxp3 was reduced in CD4+/CD25- and in CD4+/CD25+ cells and ICOS was reduced in CD4+/CD25+ cells but it was increased in CD4+CD25-in asthmatics when compared to controls. In asthmatics, in vitro, budesonide was able to: 1) increase annexin V binding and to reduce ICOS in total lymphocytes; 2) increase annexin V binding and Foxp3 and to reduce ICOS in CD4+/CD25- cells; 3) reduce annexin V binding and to increase IL-10 and ICOS in CD4+/CD25+ cells; 4) reduce cell allergen induced proliferation. In vivo, budesonide increased ICOS in CD4+/CD25+ while it increased Foxp3 and IL-10 in CD4+/CD25+ and in CD4+/CD25- cells. Conclusions Budesonide modulates T cell survival, ICOS, Foxp3 and IL-10 molecules differently in T lymphocyte sub-populations. The findings provided shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation. PMID:23251336

  14. Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism

    PubMed Central

    Labiano, Sara; Palazón, Asis; Bolaños, Elixabet; Azpilikueta, Arantza; Sánchez-Paulete, Alfonso R.; Morales-Kastresana, Aizea; Quetglas, Jose I.; Perez-Gracia, José L.; Gúrpide, Alfonso; Rodriguez-Ruiz, Maria; Aznar, M. Angela; Jure-Kunkel, Maria; Berraondo, Pedro; Melero, Ignacio

    2016-01-01

    ABSTRACT Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8+ T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L. PMID:26942078

  15. Absence of histophatological response to cadmium in gill and digestive diverticula of the mussel, Mytilus edulis

    SciTech Connect

    Giraud, A.S.; Webster, L.K.; Fabris, J.G.; Collett, L.C.; Yeomans, N.D.

    1986-01-01

    The blue mussel (Mytilus edulis) has been proposed for use as a sentinel organism to monitor the effects of marine pollution (Goldberg et al., 1978). Recently, there has been interest in quantifying histopathological changes in mussel tissues, as one indicator of pollution-induced stress. Cadmium is a common and toxic aquatic pollutant. Gill and digestive diverticula have been shown to be major sites of cadmium detoxification. In these same tissues, histopathological changes have been demonstrated after exposure to crude oil and to an oil dispersant. However, whether similar morphological changes are induced by heavy metals, such as cadmium, is not known. In this study, the authors have assessed the cellular effects of sublethal concentrations of cadmium on the gill and digestive diverticula of Mytilus.

  16. Recurrent amoebic gill infestation in rainbow trout cultured in a semiclosed water recirculation system

    USGS Publications Warehouse

    Noble, A.C.; Herman, R.L.; Noga, E.J.; Bullock, G.L.

    1997-01-01

    Five lots of commercially purchased juvenile rainbow trout Oncorhynchus mykiss (17-44 g) stocked in a continuous-production water recirculation system became infested with gilt amoebae. The amoebae were introduced into the recirculation system, as evidenced by their presence on gills of fish held in quarantine tanks. Based on their morphology, as seen in histological sections and by electron microscopy, the amoebae appeared to be more closely related to the family Cochliopodiidae than to other taxa of free living amoebae. Attempts to culture the amoebae in different media, at different temperatures of incubation, and in fish cell culture were not successful. Initial treatment of the recirculation system with formalin at 167 parts per million (ppm) for 1 h eliminated amoebae from the gills. Subsequent treatments of the entire system with formalin at 50-167 ppm reduced the intensity of further infestations.

  17. Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

    PubMed

    Paniagua, E; Paramá, A; Iglesias, R; Sanmartín, M L; Leiro, J

    2001-05-04

    We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.

  18. In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

    NASA Astrophysics Data System (ADS)

    Su, Feng; Zhang, Shicui; Li, Hongyan; Guo, Huarong

    2007-04-01

    In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR. MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.

  19. Experimental lead nitrate poisoning: microscopic and ultrastructural study of the gills of tench (Tinca tinca, L.).

    PubMed Central

    Roncero, V; Vincente, J A; Redondo, E; Gãzquez, A; Duran, E

    1990-01-01

    A microscopic, ultrastructural, and morphometric study was made of the gills of tench (Tinca tinca, L.) subjected to acute experimental lead nitrate poisoning. Twenty-one adult tench were subjected to poisoning and a further 22 were used as controls. Lesions were characterized by the appearance of edema and epithelial hyperplasia and necrosis, both in cells forming part of the filtration barrier and in those in the interlamellar space. These processes developed in the course of the experiment, leading to the death of tench after 12 to 15 days of exposure to 75 ppm lead nitrate, at which point the concentrations of lead in the gills had reached their maximum. Images FIGURE 1. FIGURE 2. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12. FIGURE 13. FIGURE 14. FIGURE 15. FIGURE 16. FIGURE 17. FIGURE 18. FIGURE 19. PMID:2088740

  20. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

    PubMed Central

    Alarcón, B; Ley, S C; Sánchez-Madrid, F; Blumberg, R S; Ju, S T; Fresno, M; Terhorst, C

    1991-01-01

    The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development. Images PMID:1826255

  1. The metabolic effects of limbic leucotomy in Gilles de la Tourette syndrome.

    PubMed Central

    Sawle, G V; Lees, A J; Hymas, N F; Brooks, D J; Frackowiak, R S

    1993-01-01

    Regional cerebral oxygen metabolism was measured before and after limbic leucotomy in a patient with Gilles de la Tourette syndrome, obsessive compulsive disorder, and obsessional slowness. The preoperative scan showed hypermetabolism in the caudate nuclei, which normalised after operation. It is proposed that the beneficial effects of this operation on both tics and obsessive compulsive behaviour are mediated by disruption of abnormal neural activity in basal ganglia-thalamocortical loops. Images PMID:8410025

  2. Effects of Potential Therapeutic Agents on Copper Accumulations in Gill of Crassostrea virginica

    PubMed Central

    Luxama, Juan D.; Carroll, Margaret A.; Catapane, Edward J.

    2010-01-01

    Copper is an essential trace element for organisms, but when in excess, copper’s redox potential enhances oxyradical formation and increases cellular oxidative stress. Copper is a major pollutant in Jamaica Bay and other aquatic areas. Bivalves are filter feeders that accumulate heavy metals and other pollutants from their environment. Previously it was determined that seed from the bivalve Crassostrea virginica, transplanted from an oyster farm to Jamaica Bay readily accumulated copper and other pollutants into their tissues. In the present study we utilized Atomic Absorption Spectrometry to measure the uptake of copper into C. virginica gill