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Sample records for gill cd binding

  1. Modeling silver binding to gills of rainbow trout (Oncorhynchus mykiss)

    SciTech Connect

    Janes, N.; Playle, R.C.

    1995-11-01

    Rainbow trout (Oncorhynchus mykiss, 1--3 g) were exposed to {approximately} 1.0 {micro}M silver (Ag) ({approximately} 11 {micro}g {center_dot} L{sup {minus}1} Ag) for 2 to 3 h in synthetic soft water (Ca, Na {approximately} 300 {micro}M, pH 6.5--7.5) to which was added Ca, Na, H{sup +}, dissolved organic carbon (DOC), Cl, or thiosulfate (S{sub 2}O{sub 3}). Gills were extracted and gill Ag concentrations were measured using graphite-furnace atomic absorption spectrophotometry. The concentration of cations (Ca, Na, H{sup +}) and complexing agents (DOC, Cl, S{sub 2}O{sub 3}) needed to keep Ag off the gills were used to calculate conditional equilibrium binding constants (K) at the gills. Log K for Ag-gill binding was 10.0, with approximately 1.3 nmol Ag binding sites per fish. All experimentally determined log K values were entered into an aquatic chemistry equilibrium model, MINEQL{sup +}, to predict Ag binding at trout gills. For a series of natural waters, model-predicted gill Ag concentrations correlated well with observed gill Ag concentrations, with one exception, very hard city of Waterloo tapwater. This exception may indicate a kinetic constraint on the thermodynamic basis of the model.

  2. 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

  3. 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

  4. Binding of the angiotensin-converting enzyme inhibitor, RACX-65, to trout gills

    SciTech Connect

    Olson, K.R.; Evan, A.P.; Ryan, J.W.

    1986-03-01

    Galardy et al. have shown that in rainbow trout, Salmo gairdneri, nearly all of the angiotensin converting enzyme (ACE) activity is found in the gills. In the present study, binding and localization of the angiotensin-converting enzyme inhibitor N-(1(S)-carboxanili-dopropyl)-L-Ala-L-Pro (RAC-X-65) to trout gill was examined in homogenized gill tissues, an isolated perfused gill and by autoradiography. RAC-X-65 inhibited gill ACE as effectively as it inhibits human ACE; the apparent Ki for gill homogenates fell over 15 min from 3 x 10/sup -9/M to 5.5 x 10/sup -10/M. In the arterioarterial pathway (supplying the systematic circulation) of the perfused gill, /sup 3/H-RAC-X-65 extraction (compared to the inert volume maker, /sup 14/C-sucrose) decreased from 72.2 +/- 4.5% after 6 min perfusion to 54.7 +/- 6.8% (14 min) and 38.4 +/- 9.0% after 20 min (N = 6). By 40 min, extraction was less then 10% indicating saturation of ACE binding sites. Relatively little extraction was observed in the gill arteriovenous pathway. Autoradiography of gills perfused with /sup 3/H-RAC-X-65 demonstrated that the pillar cells are the major site of /sup 3/H accumulation and, therefore, probably contain most of the ACE activity.

  5. Altered perceptual binding in Gilles de la Tourette syndrome.

    PubMed

    Beste, Christian; Tübing, Jennifer; Seeliger, Helen; Bäumer, Tobias; Brandt, Valerie; Stock, Ann-Kathrin; Münchau, Alexander

    2016-10-01

    Gilles de la Tourette syndrome (GTS) is a common multifaceted neuropsychiatric disorder. Research in GTS has traditionally focussed on mechanisms underlying changes in motor processes in these patients. There is, however, growing interest in GTS related sensory phenomena. According to the Theory of Event-coding (TEC), sensory stimuli do not only serve representational functions but also action-related functions. In the current study, we use the TEC framework to examine whether the way perceptual features are processed is altered in GTS. The results show that basic perceptual processes differ between GTS patients and healthy controls which might be central for the understanding of this disorder. Details or features of perceptual objects were less bound in GTS suggesting that perceptual features integration is attenuated in them. Behavioural findings were unrelated to patient characteristics implying that they might represent trait abnormalities. It is possible that altered perceptual processing in GTS is due to a long-range under-connectivity of parietal areas with other brain regions repeatedly been described in these patients.

  6. Altered perceptual binding in Gilles de la Tourette syndrome.

    PubMed

    Beste, Christian; Tübing, Jennifer; Seeliger, Helen; Bäumer, Tobias; Brandt, Valerie; Stock, Ann-Kathrin; Münchau, Alexander

    2016-10-01

    Gilles de la Tourette syndrome (GTS) is a common multifaceted neuropsychiatric disorder. Research in GTS has traditionally focussed on mechanisms underlying changes in motor processes in these patients. There is, however, growing interest in GTS related sensory phenomena. According to the Theory of Event-coding (TEC), sensory stimuli do not only serve representational functions but also action-related functions. In the current study, we use the TEC framework to examine whether the way perceptual features are processed is altered in GTS. The results show that basic perceptual processes differ between GTS patients and healthy controls which might be central for the understanding of this disorder. Details or features of perceptual objects were less bound in GTS suggesting that perceptual features integration is attenuated in them. Behavioural findings were unrelated to patient characteristics implying that they might represent trait abnormalities. It is possible that altered perceptual processing in GTS is due to a long-range under-connectivity of parietal areas with other brain regions repeatedly been described in these patients. PMID:27544346

  7. 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.

  8. 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.

  9. 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. PMID:27003804

  10. Effects of copper and cadmium on ion transport and gill metal binding in the Amazonian teleost tambaqui (Colossoma macropomum) in extremely soft water.

    PubMed

    Matsuo, Aline Y O; Wood, Chris M; Val, Adalberto L

    2005-09-30

    Metal toxicity in fish is expected to be most severe in soft waters because of the low availability of cations (particularly Ca(2+)) to out-compete the metal forms for binding sites on the gills. Natural waters in the Amazon basin are typically soft due to regional geochemistry, but few studies have focused on metal toxicity in fish native to the basin. We assessed the ionoregulatory effects of waterborne copper (Cu) and cadmium (Cd) on tambaqui (Colossoma macropomum) in extremely soft water (10 micromoll(-1) Ca(2+)). Tambaqui had a very high tolerance to Cu (50-400 microgl(-1)), as indicated by a complete lack of inhibition of Na(+) uptake and an ability to gradually recover over 6h from elevated diffusive Na(+) losses caused by Cu. The insensitivity of active Na(+) influx to Cu further supports the notion that Amazonian fish may have a unique Na(+) transport system. Addition of 5-10 mgCl(-1) of dissolved organic matter (DOM) did not prevent initial (0-3h) negative Na(+) balance in tambaqui exposed to Cu. Exposure to 40 mgCl(-1) DOM prevented Na(+) losses in tambaqui even at 400 microgl(-1) Cu, probably because most Cu was complexed to DOM. Tambaqui exposed to waterborne Cd (10-80 microgl(-1)) experienced an average of 42% inhibition in whole body Ca(2+) uptake relative to controls within 3h of exposure to the metal. Inhibition of Ca(2+) uptake increased over time and, at 24h, Ca(2+) uptake was suppressed by 51% and 91% in fish exposed to 10 and 80 microgl(-1) Cd, respectively. Previous acclimation of fish to either elevated [Ca(2+)] or elevated [DOM] proved to be very effective in protecting against acute short-term metal accumulation at the gills of tambaqui in soft water (in the absence of the protective agent during metal exposure), suggesting a conditioning effect on gill metal binding physiology. PMID:16051381

  11. 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

  12. 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.

  13. 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

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

    PubMed

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

    2016-01-15

    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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Zinc is essential for binding of p56(lck) to CD4 and CD8alpha.

    PubMed

    Lin, R S; Rodriguez, C; Veillette, A; Lodish, H F

    1998-12-01

    Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.

  20. Subcellular localization of Cd and Cd-binding peptides in tobacco leaves

    SciTech Connect

    Vogeli-Lange, R.; Wagner, G.J. )

    1989-04-01

    Cd-binding peptides (CdBP's) having the general structure {gamma}-(Glu-Cys){sub n}-Gly are inducible by and have high affinity for Cd. If these peptides are involved in Cd detoxification by chelation, both metal and ligand must be localized in the same cellular compartment. To address this question, we studied the vacuolar/extravacuolar distribution of Cd and CdBP's in leaves of hydroponically grown tobacco seedlings. CdBP's were induced upon addition of 20 {mu}M CdCl{sub 2} (non-phytotoxic level) to the nutrient solution. Amino acid analysis indicated that the main components were {gamma}-(Glu-Cys){sub 3}-Gly and {gamma}-(Glu-Cys){sub 4}-Gly. Purified vacuoles isolated from protoplasts of Cd treated leaves contained most of the total CdBP's and Cd found in protoplasts (104% {plus minus}8 and 110% {plus minus}8, respectively). The probability that CdBP's are synthesized extravacuolarly and their predominant location in the vacuole suggest that these molecules may be involved in translocation of Cd to the vacuole.

  1. Isolation and characterization of a novel fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis).

    PubMed

    Pan, Saikun; Tang, Jian; Gu, Xiaohong

    2010-02-15

    Lectins play an important role in many biological systems and are used increasingly in human disease therapy. To improve our understanding of fish lectins we purified and characterized a fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis). The purification procedure consisted of extracting soluble proteins in 25mM Tris-HCl buffer (pH 8.5), separation on a DEAE-Sepharose FF ion exchange column, followed by gel filtration chromatography on Sephacryl S-200 HR and Superdex 200 10/300 GL columns. The purified lectin, designated GANL, had a single protein band with an apparent molecular mass of 37kDa when subject to SDS-PAGE under reducing conditions. GANL is a homomultimeric glycoprotein with a native molecular mass of 220kDa and a carbohydrate content of approximately 13.4%. The purified lectin only agglutinated rabbit native erythrocytes, and did not require Ca(2+). Its activity was not inhibited by any of the mono- or disaccharides or glycoproteins tested, except for fucose. GANL contains a high proportion of Asp, Glu, Leu, Val, and Lys. The first 10 residues of the N-terminal region were determined as AGEQGGQCSA. The anti-microbial activity was assessed by measuring agglutination and inhibition of pathogen growth. Our results suggest that GANL agglutinates and inhibits the growth of Vibrio harveyi but has no antifungal activity. PMID:19709756

  2. Prolactin receptors in liver, kidney, and gill of the tilapia (Oreochromis mossambicus): Characterization and effect of salinity on specific binding of iodinated ovine prolactin

    SciTech Connect

    Dauder, S.; Young, G.; Hass, L.; Bern, H.A. )

    1990-03-01

    Specific binding of {sup 125}I-ovine prolactin (oPRL) to microsomal fractions from gill, kidney, and liver of adult tilapia was determined. Specific binding varied among tissues, the highest values being displayed by kidney membranes. In the liver, the binding of oPRL was not strongly displaced by tilapia prolactins (tPRL177 and tPRL188), although tPRL177 was six times more potent than tPRL188. On the other hand, in kidney and gill membranes, the two tPRLs were equipotent. Tilapia PRLs showed low potency in competing for oPRL-binding sites when pregnant rat liver membranes were utilized. Tilapia growth hormone (tGH) and human growth hormone (hGH) displaced {sup 125}I-oPRL from liver as well as did tPRL177 but were not recognized well by renal or branchial receptors. Two {sup 125}I-oPRL-binding sites were detected in every tissue tested. These binding sites are subject to physiological regulation since adaptation to seawater resulted in a significant decrease in specific binding.

  3. 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.

  4. Energetics of the HIV gp120-CD4 binding reaction.

    PubMed

    Myszka, D G; Sweet, R W; Hensley, P; Brigham-Burke, M; Kwong, P D; Hendrickson, W A; Wyatt, R; Sodroski, J; Doyle, M L

    2000-08-01

    HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

  5. IL-15 prolongs CD154 expression on human CD4 T cells via STAT5 binding to the CD154 transcriptional promoter

    PubMed Central

    Lowe, RM; Genin, A; Orgun, N; Cron, RQ

    2014-01-01

    Activation induced CD154 expression on CD4 T cells is prolonged in systemic lupus erythematosus but the mechanism(s) for its dysregulation are unknown. The studies reported herein demonstrate that IL-15 is capable of prolonging CD154 expression on PHA activated CD4 T cells. Since IL-15 signals through STAT5, predicted STAT5 binding sites in the human CD154 transcriptional promoter were identified, and STAT5 binding to the proximal CD154 promoter in vitro and in vivo following primary CD4 T cell activation was demonstrated. Moreover, overexpression of wild-type(WT) STAT5 in primary human CD4 T cells augmented CD154 transcription, whereas overexpression of a dominant negative (DN) STAT5 protein inhibited CD154 transcription. Mutation of the most proximal STAT5 binding site in the CD154 promoter resulted in diminished DNA binding and reduced CD154 transcriptional activity. Interestingly, STAT5-specific siRNA inhibited CD154 surface expression at 48 but not 24 hours after T cell activation. Thus, these findings provide some of the first evidence to support a possible mechanistic link to explain how the overexpression of IL-15 observed in lupus patients may be involved in the prolonged expression of CD154 that has also been observed on lupus CD4 T cells. PMID:24500400

  6. Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands

    PubMed Central

    Yi, Ling; Zhao, Yanlin; Wang, Xiaojue; Dai, Min; Hellström, Karl Erik; Hellström, Ingegerd; Zhang, Hongtao

    2014-01-01

    Monoclonal antibodies (mAbs) to CD137 (a.k.a. 4-1BB) have anti-tumor efficacy in several animal models and have entered clinical trials in patients with advanced cancer. Importantly, anti-CD137 mAbs can also ameliorate autoimmunity in preclinical models. As an approach to better understand the action of agonistic and antagonistic anti-CD137 mAbs we have mapped the binding region of the CD137 ligand (CD137L) to human and mouse CD137. By investigating the binding of CD137L to cysteine rich domain II (CRDII )and CRDIII of CD137, we found that the binding interface was limited and differed between the two species in that mouse CD137L mainly combined with CRDII and human CD137L mainly combined with CRDIII. PMID:24466035

  7. 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.

  8. Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells

    PubMed Central

    Shiratsuchi, Takayuki; Schneck, Jonathan; Kawamura, Akira; Tsuji, Moriya

    2009-01-01

    Antigen presenting molecules play an important role in both innate and adoptive immune responses by priming and activating T cells. Among them, CD1 molecules have been identified to present both exogenous and endogenous lipid antigens to CD1-restricted T cells. The involvement of CD1-restricted T cells in autoimmune diseases and in defense against infectious diseases, however, remains largely unknown. Identifying novel antigenic lipids that bind to CD1 molecules and understanding the role of CD1-restricted T cells should lead to the successful development of vaccines, because the lipids can be used as antigens and also as adjuvants. In this paper, we have constructed functional recombinant human CD1 dimeric proteins and established a competitive ELISA assay to measure the lipid binding to CD1 molecules using the CD1 dimers. By using the competitive ELISA assay, we were able to show that the lipid extracts from murine malaria parasites can actually be loaded onto CD1 molecules. In addition, we have demonstrated that artificial antigen-presenting cells, which consist of magnetic beads coated with CD1d dimer and anti-CD28 antibody, stimulated and expanded human invariant NKT cells as efficiently as autologous immature DCs. A set of the tools presented in the current study should be valuable for screening various CD1 molecule-binding lipid antigens and for isolating CD1-restricted T cells. PMID:19374905

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26181364

  10. Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid.

    PubMed

    Ahrens, T; Sleeman, J P; Schempp, C M; Howells, N; Hofmann, M; Ponta, H; Herrlich, P; Simon, J C

    2001-06-01

    Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo. PMID:11423990

  11. The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

    PubMed Central

    Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

    2000-01-01

    Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

  12. Unique properties of cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Hayashi, Y.; Nakagawa, C.W.; Murasugi, A.

    1986-03-01

    Metallothioneins, a class of low molecular weight cysteine-rich proteins that bind heavy metal ions, have been found in various eucaryotic organisms. When fission yeasts are grown in the presence of high concentration of CdCl/sub 2/, large amounts of Cd-binding peptides (Cd-BP1 and Cd-BP2) are synthesized. While Cd-BP2 shows similarities to mammalian Cd-thioneins in UV and CD spectra, Cd-BP1has a characteristic shoulder at 265 nm in the UV absorption spectrum and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These characteristics of Cd-BP1 are not found in the other Cd-thioneins. The UV and CD spectra differences between reconstituted and native Cd-BP1 suggest the requirement for some additional molecular architecture including another peptide-Cd/sup 2 +/ interaction. Induction of cadystin synthesis is almost exclusive for Cd, but an exception is a small amount of cadystin also induced by the higher concentration of CuCl/sub 2/ (2.5 mM). The UV spectrum of the natural Cu-cadystin complex was similar to that of Cd-BP1. On the basis of these findings the models for Cd-BP1 and Cd-BP2 are proposed.

  13. 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

  14. Induced conformational changes upon Cd2+ binding at photosynthetic reaction centers

    PubMed Central

    Ishikita, Hiroshi; Knapp, Ernst-Walter

    2005-01-01

    Cd2+ binding at the bacterial photosynthetic reaction center (bRC) from Rhodobacter sphaeroides is known to inhibit proton transfer (PT) from bulk solvent to the secondary quinone QB. To elucidate this mechanism, we calculated the pKa for residues along the water channels connecting QB with the stromal side based on the crystal structures of WT-bRC and Cd2+-bound bRC. Upon Cd2+ binding, we observed the release of two protons from His-H126/128 at the Cd2+ binding site and significant pKa shifts for residues along the PT pathways. Remarkably, Asp-L213 near QB, which is proposed to play a significant role in PT, resulted in a decrease in pKa upon Cd2+ binding. The direct electrostatic influence of the Cd2+-positive charge on these pKa shifts was small. Instead, conformational changes of amino acid side chains induced electrostatically by Cd2+ binding were the main mechanism for these pKa shifts. The long-range electrostatic influence over ≈12 Å between Cd2+ and Asp-L213 is likely to originate from a set of Cd2+-induced successive reorientations of side chains (Asp-H124, His-H126, His-H128, Asp-H170, Glu-H173, Asp-M17, and Asp-L210), which propagate along the PT pathways as a “domino” effect. PMID:16254054

  15. Reductions of Cd in a Cd-contaminated fish by long-term exposure to EDTA or fresh-water

    SciTech Connect

    Muramoto, S.

    1982-01-01

    The variations in Cd levels with the passage of time in the viscera and gills of carp (Cyprinus carpio L.) after exposure to 0.01 and 0.05 ppm Cd containing water for 3 months were determined. After 3 months, the fish were treated with fresh-water. The fresh-water treatment produced slow recovery in the viscera, but the Cd concentration in the gills remained approximately the same. From the results of the study, it was postulated that the recovery of fish contaminated with Cd and other heavy metals would seem possible due to loose bindings of the metals to protein of the fish. (JMT)

  16. CD36 mediates proximal tubular binding and uptake of albumin and is upregulated in proteinuric nephropathies.

    PubMed

    Baines, Richard J; Chana, Ravinder S; Hall, Matthew; Febbraio, Maria; Kennedy, David; Brunskill, Nigel J

    2012-10-01

    Dysregulation of renal tubular protein handling in proteinuria contributes to the development of chronic kidney disease. We investigated the role of CD36 as a novel candidate mediator of albumin binding and endocytosis in the kidney proximal tubule using both in vitro and in vivo approaches, and in nephrotic patient renal biopsy samples. In CD36-transfected opossum kidney proximal tubular cells, both binding and uptake of albumin were substantially enhanced. A specific CD36 inhibitor abrogated this effect, but receptor-associated protein, which blocks megalin-mediated endocytosis of albumin, did not. Mouse proximal tubular cells expressed CD36 and this was absent in CD36 null animals, whereas expression of megalin was equal in these animals. Compared with wild-type mice, CD36 null mice demonstrated a significantly increased urinary protein-to-creatinine ratio and albumin-to-creatinine ratio. Proximal tubular cells expressed increased CD36 when exposed to elevated albumin concentrations in culture medium. Expression of CD36 was studied in renal biopsy tissue obtained from adult patients with heavy proteinuria due to minimal change disease, membranous nephropathy, or focal segmental glomerulosclerosis. Proximal tubular CD36 expression was markedly increased in proteinuric individuals. We conclude that CD36 is a novel mediator influencing binding and uptake of albumin in the proximal tubule that is upregulated in proteinuric renal diseases. CD36 may represent a potential therapeutic target in proteinuric nephropathy. PMID:22791331

  17. Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

    PubMed

    Armitage, Ian M; Drakenberg, Torbjörn; Reilly, Brian

    2013-01-01

    Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd

  18. 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.

  19. 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

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

    PubMed

    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.

  1. MiniCD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency

    PubMed Central

    2012-01-01

    Background Binding of the viral envelope protein (Env), and particularly of its gp120 subunit, to the cellular CD4 receptor is the first essential step of the HIV-1 entry process. The CD4 binding site (CD4bs) of gp120, and especially a recessed cavity occupied by the CD4 Phe43 residue, are known to be highly conserved among the different circulating subtypes and therefore constitute particularly interesting targets for vaccine and drug design. The miniCD4 proteins are a promising class of CD4bs inhibitors. Studying virus evolution under pressure of CD4bs inhibitors could provide insight on the gp120-CD4 interaction and viral entry. Results The present study reports on the resistance induction of two subtype B HIV-1 against the most active miniCD4, M48U1, and its ancestor, M48, and how these mutated positions affect CD4bs recognition, entry efficiency, and sensitivity to other CD4bs inhibitors. Resistance against M48U1 was always associated with S375R/N substitution in both BaL and SF162; M48 resistance was associated with D474N substitution in SF162 and with H105Y substitution in BaL. In addition, some other mutations at position V255 and G471 were of importance for SF162 resistant viruses. Except for 474, all of these mutated positions are conserved, and introducing them into an SF162 Env expressing infectious molecular clone (pBRNL4.3 SF162) resulted in decreased entry efficiency. Furthermore, resistant mutants showed at least some cross-resistance towards other CD4bs inhibitors, the V3 monoclonal antibody 447-52D and some even against the monoclonal antibody 17b, of which the epitope overlaps the co-receptor binding site. Conclusions The mutations H105Y, V255M, S375R/N, G471R/E, and D474N are found to be involved in resistance towards M48 and M48U1. All mutated positions are part of, or in close proximity to, the CD4bs; most are highly conserved, and all have an impact on the entry efficiency, suggesting their importance for optimal virus infectivity. PMID

  2. Ligand binding to anti-cancer target CD44 investigated by molecular simulations.

    PubMed

    Nguyen, Tin Trung; Tran, Duy Phuoc; Pham Dinh Quoc Huy; Hoang, Zung; Carloni, Paolo; Van Pham, Phuc; Nguyen, Chuong; Li, Mai Suan

    2016-07-01

    CD44 is a cell-surface glycoprotein and receptor for hyaluronan, one of the major components of the tumor extracellular matrix. There is evidence that the interaction between CD44 and hyaluronan promotes breast cancer metastasis. Recently, the molecule F-19848A was shown to inhibit hyaluronan binding to receptor CD44 in a cell-based assay. In this study, we investigated the mechanism and energetics of F-19848A binding to CD44 using molecular simulation. Using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) method, we obtained the binding free energy and inhibition constant of the complex. The van der Waals (vdW) interaction and the extended portion of F-19848A play key roles in the binding affinity. We screened natural products from a traditional Chinese medicine database to search for CD44 inhibitors. From combining pharmaceutical requirements with docking and molecular dynamics simulations, we found ten compounds that are potentially better or equal to the F-19848A ligand at binding to CD44 receptor. Therefore, we have identified new candidates of CD44 inhibitors, based on molecular simulation, which may be effective small molecules for the therapy of breast cancer. PMID:27342250

  3. 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.

  4. Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob.

    PubMed

    Wang, Hongjie; Liaw, Yen-Chywan; Stone, Daniel; Kalyuzhniy, Oleksandr; Amiraslanov, Imameddin; Tuve, Sebastian; Verlinde, Christophe L M J; Shayakhmetov, Dmitry; Stehle, Thilo; Roffler, Steve; Lieber, André

    2007-12-01

    Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a KD (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-A resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.

  5. Catalytic properties of lipopolysaccharide (LPS) binding protein. Transfer of LPS to soluble CD14.

    PubMed

    Yu, B; Wright, S D

    1996-02-23

    Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS.sCD14 complexes to HDL particles. To characterize the first of these two reactions, LPS covalently derivatized with the fluorophore, boron dipyrromethene difluoride (BODIPY), was used to monitor LBP-catalyzed movement of LPS in real time. The fluorescence efficiency of micelles of BODIPY-LPS was low but was strongly increased upon dissolution in detergent or upon binding to sCD14. Spontaneous binding of BODIPY-LPS to sCD14 was very slow but was accelerated by substoichiometric concentration of LBP, and the rate of binding was measured under a variety of conditions. LBP-catalyzed transfer was first order with respect to both sCD14 and LPS concentration, and the apparent Km values were 1 approximately 2 microg/ml for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP was approximately 150 molecules of LPS min-1 LBP-1. LBP alone caused a small but measurable increase in the fluorescence of BODIPY-LPS, suggesting that it bound LPS aggregates but did not readily remove LPS monomers. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14. These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate. PMID:8626747

  6. Crystal structure of two CD46 domains reveals an extended measles virus-binding surface.

    PubMed Central

    Casasnovas, J M; Larvie, M; Stehle, T

    1999-01-01

    Measles virus is a paramyxovirus which, like other members of the family such as respiratory syncytial virus, is a major cause of morbidity and mortality worldwide. The cell surface receptor for measles virus in humans is CD46, a complement cofactor. We report here the crystal structure at 3.1 A resolution of the measles virus-binding fragment of CD46. The structure reveals the architecture and spatial arrangement of two glycosylated short consensus repeats with a pronounced interdomain bend and some flexibility at the domain interface. Amino acids involved in measles virus binding define a large, glycan-free surface that extends from the top of the first to the bottom of the second repeat. The extended virus-binding surface of CD46 differs strikingly from those reported for the human virus receptor proteins CD4 and intercellular cell adhesion molecule-1 (ICAM-1), suggesting that the CD46 structure utilizes a novel mode of virus recognition. A highly hydrophobic and protruding loop at the base of the first repeat bears a critical virus-binding residue, thereby defining an important recognition epitope. Molecules that mimic the conformation of this loop potentially could be effective anti-viral agents by preventing binding of measles virus to CD46. PMID:10357804

  7. Characterization of H+ and Cd2+ binding properties of the bacterial exopolysaccharides.

    PubMed

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

    2006-11-01

    The proton and Cd binding capacities of microbially produced exopolysaccharides, EPS, were quantified by the determination of stability constants and the concentration of complexing sites using H(+) or Cd(2+) selective electrodes in dynamic titrations. The influence of ionic strength, pH and the Cd to EPS ratio was evaluated over large concentration ranges. The applicability of the non-ideal competitive adsorption isotherm combined with a Donnan electrostatics approach was tested with respect to the EPS. Proton and cadmium binding data were compared with literature data examining other ubiquitous environmental ligands including humic substances, alginate, bacteria, etc. Subsequent modelling of Cd speciation in aquatic (fresh and marine waters) and soil systems suggested that the exopolysaccharides would play non-negligible role, under most conditions. The quantitative information provided in this paper thus represents an important advance in our understanding of Cd transport, bioavailability and impact in aquatic and terrestrial systems. PMID:16753198

  8. 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.

  9. Soluble CD109 binds TGF-β and antagonizes TGF-β signalling and responses.

    PubMed

    Li, Carter; Hancock, Mark A; Sehgal, Priyanka; Zhou, Shufeng; Reinhardt, Dieter P; Philip, Anie

    2016-03-01

    Transforming growth factor-β (TGF-β) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-β mediates diverse biological responses by signalling through type I and II TGF-β receptors (TβRI and TβRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-β co-receptor that negatively regulates TGF-β signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-β receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-β antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-β and inhibit TGF-β signalling and responses. Our results demonstrate that sCD109 binds TGF-β with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-β at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-β-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-β, inhibit TGF-β binding to its receptors and decrease TGF-β signalling and TGF-β-induced cellular responses.

  10. Design and syntheses of hyaluronan oligosaccharide conjugates as inhibitors of CD44-Hyaluronan binding

    PubMed Central

    Huang, Xuefei

    2016-01-01

    Hyaluronan (HA) is an integral component of the extracellular matrix. Its interactions with a cell surface receptor CD44 has been shown to play important roles in a variety of biological events including cell proliferation and metastasis. As multivalent CD44-HA binding is critical for downstream signaling, compounds that can selectively disrupt the complex formation of HA polysaccharide with CD44 can serve as useful probes of CD44 mediated cellular events as well as potential leads for novel therapeutics. Herein, we report the synthesis of several series of HA conjugates to target the HA binding pocket of CD44. As a small library of HA disaccharide derivatives failed to exhibit any inhibitory activities, we focused on HA tetrasaccharide based analogs. Traditional synthetic strategies towards HA oligosaccharides involve the construction of backbone from the corresponding monosaccharide building blocks, which can be quite tedious. In order to expedite the synthesis, we designed a new synthetic route taking advantage of the ability of hyaluronidase to generate large quantities of HA tetrasaccharide through digestion of HA polysaccharides. The HA tetrasaccharide obtained was utilized to prepare multiple S-linked HA analogs bearing aromatic groups at the reducing end glycan. One such compound containing an m-benzyl phenyl moiety exhibited significant inhibition of CD44-HA binding. Our approach provides a new direction towards the design of HA based CD44 antagonists. PMID:25997408

  11. 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

  12. Zebrafish CD59 has both bacterial-binding and inhibiting activities.

    PubMed

    Sun, Chen; Wu, Jie; Liu, Shousheng; Li, Hongyan; Zhang, Shicui

    2013-10-01

    CD59, known as protectin, usually plays roles as a regulatory inhibitor of complement, but it also exhibits activities independent of its function as a complement inhibitor. This study reported the identification and characterization of an ortholog of mammalian cd59 from zebrafish Danio rerio, which is similar to known cd59 in terms of both amino acid sequence and genomic structure as well as synteny conservation. We showed that zebrafish cd59 was maternally expressed in early embryos and expressed in a tissue-specific manner, with most abundant expression in the brain. We further showed that recombinant zebrafish CD59 was capable of binding to both the Gram-negative and Gram-positive bacteria as well as the microbial signature molecules LPS and LTA. In addition we demonstrated that recombinant zebrafish CD59 displayed slight antimicrobial activity capable of inhibiting the growth of E. coli and S. aureus. All these data indicate that zebrafish CD59 can not only binds to the bacteria and their signature molecules LPS and LTA but can also inhibit their growth, a novel role assigned to CD59.

  13. Cadmium-binding protein (metallothionein) in carp

    SciTech Connect

    Kito, H.; Ose, Y.; Sato, T.

    1986-03-01

    When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl/sub 2/), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups.

  14. 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.

  15. 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

  16. 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

  17. Quantitative measurements of magnetic polaron binding on acceptors in CdMnTe alloys

    NASA Astrophysics Data System (ADS)

    Nhung, Tran Hong; Planel, R.

    1983-03-01

    The acceptor binding energy is measured as a function of Temperature and composition in Cd1-x Mnx Te alloys, by time resolved spectroscopy. The Bound magnetic polaron effect is measured and compared with a theory accouting for magnetic saturation and fluctuations.

  18. 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

  19. 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. PMID:21889131

  20. 13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin.

    PubMed

    Bjornson, M E; Corson, D C; Sykes, B D

    1985-10-01

    13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.

  1. CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis

    PubMed Central

    Leemans, Jaklien C.; Florquin, Sandrine; Heikens, Mirjam; Pals, Steven T.; Neut, Ronald van der; van der Poll, Tom

    2003-01-01

    Cell migration and phagocytosis are both important for controlling Mycobacterium tuberculosis infection and are critically dependent on the reorganization of the cytoskeleton. Since CD44 is an adhesion molecule involved in inflammatory responses and is connected to the actin cytoskeleton, we investigated the role of CD44 in both these processes. Macrophage (Mφ) recruitment into M. tuberculosis–infected lungs and delayed-type hypersensitivity sites was impaired in CD44-deficient (CD44–/–) mice. In addition, the number of T lymphocytes and the concentration of the protective key cytokine IFN-γ were reduced in the lungs of infected CD44–/– mice. The production of IFN-γ by splenocytes of CD44–/– mice was profoundly increased upon antigen-specific stimulation. Flow cytometry analysis revealed that soluble CD44 can directly bind to virulent M. tuberculosis. Mycobacteria also interacted with Mφ-associated CD44, as reflected by reduced binding and internalization of bacilli by CD44–/– Mφs. This suggests that CD44 is a receptor on Mφs for binding of M. tuberculosis. CD44–/– mice displayed a decreased survival and an enhanced mycobacterial outgrowth in lungs and liver during pulmonary tuberculosis. In summary, we have identified CD44 as a new Mφ binding site for M. tuberculosis that mediates mycobacterial phagocytosis, Mφ recruitment, and protective immunity against pulmonary tuberculosis. PMID:12618522

  2. Generation and characterization of a tetraspanin CD151/integrin α6β1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC

    PubMed Central

    Cai, Jia-Bin; Huang, Xiao-Yong; Wu, Chao; Zhang, Lu; Kang, Qiang; Liu, Li-Xin; Xie, Nan; Shen, Zao-Zhuo; Hu, Mei-Yu; Cao, Ya; Qiu, Shuang-Jian; Sun, Hui-Chuan; Zhou, Jian; Fan, Jia; Shi, Guo-Ming

    2016-01-01

    Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6β1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6β1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6β1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6β1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC. PMID:26756217

  3. 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

  4. Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

    PubMed Central

    Simon, J H; Stumbles, P; Signoret, N; Somoza, C; Puklavec, M; Sattentau, Q J; Barclay, A N; James, W

    1997-01-01

    CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus. PMID:8995673

  5. The Epstein-Barr virus-binding site on CD21 is involved in CD23 binding and interleukin-4-induced IgE and IgG4 production by human B cells.

    PubMed Central

    Henchoz-Lecoanet, S; Jeannin, P; Aubry, J P; Graber, P; Bradshaw, C G; Pochon, S; Bonnefoy, J Y

    1996-01-01

    Human CD21 has previously been described as a receptor for the C3d,g and iC3b proteins of complement, as a receptor for the gp350/220 envelope glycoprotein of the Epstein-Barr virus (EBV) and also as a receptor for inerferon-alpha (IFN-alpha). Structurally, CD21 consists of 15 to 16 short consensus repeats (SCR) of 60 to 75 amino acids followed by a transmembrane domain and an intracytoplasmic region. We reported that CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a new functional ligand for CD21. We recently found that the sites of interaction of CD23 on CD21 are on SCR 5 to 8 and 1-2. The first site is a lectin-sugar type of interaction and the second site is a protein-protein interaction. We report here that amongst the other ligands for CD21 (EBV, C3d,g and IFN-alpha), only EBV is able to inhibit the binding of CD23 to CD21. Furthermore, even a peptide from gp350/220 of EBV known to bind to CD21 is able to decrease CD23 binding to CD21. Since CD23/CD21 pairing is important in the control of IgE production, we tested the effect of the EBV-derived peptide on immunoglobulin production from peripheral blood mononuclear cells and purified tonsillar B cells. Interestingly, the EBV-peptide inhibited IgE and IgG4 production induced by interleukin-4, in a dose-dependent manner. The same results were obtained using either peripheral blood mononuclear cells or purified tonsillar B cells. Another CD21 ligand, C3, did not affect binding of CD23 to CD21 nor the production of IgE and IgG4. This study indicates that blocking CD23 binding to CD21 SCR 2 on human B cells selectively modulates immunoglobulin production. PMID:8707347

  6. [The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

    PubMed

    Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

    2007-01-01

    Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

  7. EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

    PubMed

    Huang, W; Wilson, G J; Brown, L J; Lam, H; Hambly, B D

    1998-10-15

    The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

  8. Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon.

    PubMed

    Maisey, Kevin; Toro-Ascuy, Daniela; Montero, Ruth; Reyes-López, Felipe E; Imarai, Mónica

    2011-12-01

    In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response. PMID:21821134

  9. Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.

    PubMed

    Schjaerff, Mette; Keller, Stefan M; Fass, Joseph; Froenicke, Lutz; Grahn, Robert A; Lyons, Leslie; Affolter, Verena K; Kristensen, Annemarie T; Moore, Peter F

    2016-03-01

    CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity. PMID:26687789

  10. Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: Comparison with the cis-fused isomers

    PubMed Central

    Dabrota, Cristian; Asim, Muhammad; Choueiri, Christine; Gargaun, Ana; Korobkov, Ilya; Butt, Ammara; Carlson, Kathryn E.; Katzenellenbogen, John A.; Wright, James S.; Durst, Tony

    2014-01-01

    Ligands which selectively activate only one of the estrogen receptors, ERα or ERβ, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERβ. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less β-selectivity than their cis-counterparts. PMID:25027938

  11. Observation of multiple, identical binding sites in the exchange of carboxylic acid ligands with CdS nanocrystals.

    PubMed

    Li, Xin; Nichols, Valerie M; Zhou, Dapeng; Lim, Cynthia; Pau, George Shu Heng; Bardeen, Christopher J; Tang, Ming L

    2014-06-11

    We study ligand exchange between the carboxylic acid group and 5.0 nm oleic-acid capped CdS nanocrystals (NCs) using fluorescence resonance energy transfer (FRET). This is the first measurement of the initial binding events between cadmium chalcogenide NCs and carboxylic acid groups. The binding behavior can be described as an interaction between a ligand with single binding group and a substrate with multiple, identical binding sites. Assuming Poissonian binding statistics, our model fits both steady-state and time-resolved photoluminescence (SSPL and TRPL, respectively) data well. A modified Langmuir isotherm reveals that a CdS nanoparticle has an average of 3.0 new carboxylic acid ligands and binding constant, Ka, of 3.4 × 10(5) M(-1).

  12. 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.

  13. 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. PMID:24489448

  14. Binding of monoclonal antibody to CD16 causes calcium mobilization in large granular lymphocytes but inhibits NK killing.

    PubMed Central

    Macintyre, E A; Wallace, D W; O'Flynn, K; Abdul-Gaffar, R; Tetteroo, P A; Morgan, G; Linch, D C

    1989-01-01

    A monoclonal antibody (mAb), CLB/FcR gran I, reactive with the CD16 Fc receptor (FcRlo/FcRIII) of human cells, leads to calcium mobilization in large granular lymphocytes (LGL) but not in granulocytes. Identical responses are obtained with F(ab')2 fragments of this antibody, indicating that the response is independent of Fc-FcR binding, and that bivalent cross-linking of this receptor is adequate for optimal calcium mobilization. The calcium response was greater in CD3- LGL compared to CD3+ LGL, although the response was augmented in the latter cells by prior rosetting with sheep red blood cells (SRBC). Calcium mobilization in CD3- LGL induced by CLB/FcR gran I is associated with inhibition of natural killer cell (NK) killing, and inhibition of the enhanced NK killing induced by the anti-CD2 low-density monoclonal antibody, 9.1. This supports the view that the NK-enhancing activity of 9.1 is due to simultaneous binding to CD2 and CD16, and may in fact be transduced through the CD16 molecule. The variable reported effects of anti-CD16 antibodies on NK killing are likely to reflect the epitope bound rather than the isotype of antibody used, since F(ab')2 fragments of CLB/FcR gran I also inhibit NK killing. PMID:2564843

  15. Variable primary coordination environments of Cd(II) binding to three helix bundles provide a pathway for rapid metal exchange.

    PubMed

    Tebo, Alison G; Hemmingsen, Lars; Pecoraro, Vincent L

    2015-12-01

    Members of the ArsR/SmtB family of transcriptional repressors, such as CadC, regulate the intracellular levels of heavy metals like Cd(II), Hg(II), and Pb(II). These metal sensing proteins bind their target metals with high specificity and affinity, however, a lack of structural information about these proteins makes defining the coordination sphere of the target metal difficult. Lingering questions as to the identity of Cd(II) coordination in CadC are addressed via protein design techniques. Two designed peptides with tetrathiolate metal binding sites were prepared and characterized, revealing fast exchange between CdS3O and CdS4 coordination spheres. Correlation of (111m)Cd PAC spectroscopy and (113)Cd NMR spectroscopy suggests that Cd(II) coordinated to CadC is in fast exchange between CdS3O and CdS4 forms, which may provide a mechanism for rapid sensing of heavy metal contaminants by this regulatory protein.

  16. 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

  17. 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

  18. Real Time Analysis of Binding between Rituximab (anti-CD20 antibody) and B Lymphoma Cells

    PubMed Central

    Tan, Liang; Lin, Peiling; Chisti, Mohammad M.; Rehman, Abdul; Zeng, Xiangqun

    2013-01-01

    CD20, expressed on greater than 90% of B-lymphocytic lymphomas, is an attractive target for antibody therapy. Rituximab is a chimeric murine/human-engineered monoclonal antibody and can selectively deplete CD20-expressing cells in peripheral blood and lymphoid tissues. The immobilization of B-lymphoblast-like Burkitt's lymphoma Raji cells on the quartz crystal microbalance (QCM) gold electrode surface using RGD tripeptide was electrochemically confirmed. The real-time processes of attachment of Raji cells on the gold electrode and the subsequent binding of Rituximab to the cells were studied using QCM biosensor. The interaction between Rituximab and Raji cells led to the increased resonant frequency shifts (Δf0) in the studied antibody concentration range from 5 to 250 µg mL−1 following the Langmuir adsorption model. From these observations, the apparent binding constant between a single-layer of Rituximab and Raji cells was calculated to be 1.6×106 M−1. Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved the specific interaction between Rituximab and B cells. The effects of Ca2+ and Mn2+ ions on the Rituximab-Raji cell interaction were also studied providing the enhanced QCM signals, in particular, further indicating that CD20 is a calcium ion channel that can transport these metal ions into the cells and accelerate the cell lysis induced by Rituximab. Thus the real time capability of QCM and its simplicity of operation are highly suitable for multipurpose studies on living cells including cell-immobilization, cytotoxicity of drugs, and the cell action mechanisms. PMID:23926879

  19. 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.

    PubMed

    Todd, Andrew S; Brinkman, Stephen; Wolf, Ruth E; Lamothe, Paul J; Smith, Kathleen S; Ranville, James E

    2009-06-01

    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 (approximately 140 mg/L as CaCO3) and soft water (approximately 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 (0-1000 microg/L) and soft water (0-250 microg/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 microg/L) and soft water (110 microg/L). Following short-term (< or =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 microg/L), with soft water gills accumulating more Zn than hard water gills. Short-term gill Zn accumulation at hard and soft water LCS0s (45-min median lethal accumulation) was similar (0.27 and 0.20 microg/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).

  20. 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.

  1. Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type.

    PubMed

    Pedersen, S A; Kristiansen, E; Andersen, R A; Zachariassen, K E

    2008-09-01

    Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.

  2. Ablation of nectin4 binding compromises CD46 usage by a hybrid vesicular stomatitis virus/measles virus.

    PubMed

    Liu, Yu-Ping; Russell, Samuel P; Ayala-Breton, Camilo; Russell, Stephen J; Peng, Kah-Whye

    2014-02-01

    Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.

  3. 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.

  4. Revealing the Mechanisms of Protein Disorder and N-Glycosylation in CD44-Hyaluronan Binding Using Molecular Simulation

    PubMed Central

    Guvench, Olgun

    2015-01-01

    The extracellular N-terminal hyaluronan binding domain (HABD) of CD44 is a small globular domain that confers hyaluronan (HA) binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA-binding site from a low affinity to a high affinity state; in the partially disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD. PMID:26136744

  5. The RNA-Binding Protein, Polypyrimidine Tract-Binding Protein 1 (PTBP1) Is a Key Regulator of CD4 T Cell Activation

    PubMed Central

    Valentín-Acevedo, Aníbal

    2016-01-01

    We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTBP1) plays a critical role in regulating the expression of CD40L in activated CD4 T cells. This is achieved mechanistically through message stabilization at late times of activation as well as by altered distribution of CD40L mRNA within distinct cellular compartments. PTBP1 has been implicated in many different processes, however whether PTBP1 plays a broader role in CD4 T cell activation is not known. To examine this question, experiments were designed to introduce shRNA into primary human CD4 T cells to achieve decreased, but not complete ablation of PTBP1 expression. Analyses of shPTB-expressing CD4 T cells revealed multiple processes including cell proliferation, activation-induced cell death and expression of activation markers and cytokines that were regulated in part by PTBP1 expression. Although there was an overall decrease in the steady-state level of several activation genes, only IL-2 and CD40L appeared to be regulated by PTBP1 at the level of RNA decay suggesting that PTBP1 is critical at different regulatory steps of expression that is gene-specific. Importantly, even though the IL-2 protein levels were reduced in cells with lowered PTBP1, the steady-state level of IL-2 mRNA was significantly higher in these cells suggesting a block at the translational level. Evaluation of T cell activation in shPTB-expressing T cells revealed that PTBP1 was linked primarily to the activation of the PLCγ1/ERK1/2 and the NF-κB pathways. Overall, our results reveal the importance of this critical RNA binding protein in multiple steps of T cell activation. PMID:27513449

  6. /sup 113/Cd NMR studies of a 1:1 Cd adduct with an 18-residue finger peptide from HIV-1 nucleic acid binding protein, p7

    SciTech Connect

    South, T.L.; Kim, B.; Summers, M.F.

    1989-01-04

    The Zn/sup 2+/ and Cd/sup 2+/ adducts with the 18-residue peptide comprising the amino acid sequence of the first finger (residues 13 through 30) of retroviral nucleic acid binding proteins p7 from HIV-1 (the causative agent of AIDS) have been prepared. /sup 1/H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The /sup 113/Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures.

  7. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.

    PubMed

    Gadola, Stephan D; Koch, Michael; Marles-Wright, Jon; Lissin, Nikolai M; Shepherd, Dawn; Matulis, Gediminas; Harlos, Karl; Villiger, Peter M; Stuart, David I; Jakobsen, Bent K; Cerundolo, Vincenzo; Jones, E Yvonne

    2006-03-20

    Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

  8. Differential regulation of monocyte cytokine release by αV and β2 integrins that bind CD23

    PubMed Central

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

    2012-01-01

    The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVβ3, αVβ5, αMβ2 and αXβ2, 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 αVβ3 or αXβ2 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, αXβ2 and αVβ3 appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMβ2 or αVβ5. PMID:22348662

  9. 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

  10. Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes.

    PubMed Central

    Bal, Nathalie; Wu, Chen Chou; Catty, Patrice; Guillain, Florent; Mintz, Elisabeth

    2003-01-01

    CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+). We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s). PMID:12383056

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

    PubMed

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

    2015-09-01

    γ-Tilmanocept ((99m)Tc-labeled-tilmanocept or [(99m)Tc]-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.

  12. Correlation of CD2 binding and functional properties of multimeric and monomeric lymphocyte function-associated antigen 3.

    PubMed

    Dustin, M L; Olive, D; Springer, T A

    1989-02-01

    LFA-3 was purified with an intact (mLFA-3) or an enzymatically removed membrane-anchoring domain (sLFA-3). Gel filtration and sucrose gradient sedimentation showed sLFA-3 to be a single highly glycosylated polypeptide chain in solution, while mLFA-3 formed micelles of 8 LFA-3 monomers. 125I-mLFA-3 bound to Jurkat T leukemic cell surface CD2 with much higher avidity than sLFA-3. mLFA-3 binding had characteristics of a multivalent interaction with cell surface CD2 and had an avidity of 1.5 nM for Jurkat cells and 12 nM for resting T cells. Two CD2 mAbs tested did not block mLFA-3 binding: 9-1 and CD2.1. These mAbs were tested in combination with LFA-3 for their ability to activate T cells. The combination of mLFA-3 and CD2.1 mAbs induced a rapid increase in cytosolic free Ca2+ in Jurkat cells which was proportional to mLFA-3 occupation of CD2 sites. sLFA-3 showed no activity in the Ca2+ flux assay. The combination of mLFA-3 and the CD2.1 mAbs significantly stimulated proliferation of PBMC. The combination of mLFA-3 and 9-1 mAbs was weakly or not mitogenic for the same cells. The combination of CD2.1 and sLFA-3 at concentrations up to 480 nM was not consistently mitogenic. Therefore, monomeric LFA-3/CD2 interaction appears to have a relatively low affinity, while multimeric LFA-3 binds with high avidity. T cell activation by binding of LFA-3 to CD2 appears to require occupation of 10(4) to 10(5) CD2 sites, which is likely to occur during adhesion, but is rare in receptor systems with soluble ligands. PMID:2463330

  13. 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

  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. 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.

  16. 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.

  17. Transferable orthogonal tight-binding parameters for ZnS and CdS

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Somesh Kr; Deodhar, Prajakta A.; Viswanatha, Ranjani; Kshirsagar, Anjali

    2010-07-01

    Calculations of Slater-Koster (SK) parameters appearing in the tight-binding method using sp3d5 basis sets for both the cationic and anionic species are presented for ZnS and CdS. We have adjusted these parameters to match the band structures obtained from the full potential linear augmented plane wave method. This operation has been carried out for a variety of structures namely zinc blende, wurtzite, rocksalt, CsCl and for a wide range of near-neighbor distances. The SK parameters have slightly different values for the same near-neighbor distance in different structures. Therefore, a least-squares fitting has been performed separately for each parameter as a function of only the near-neighbor distance to guarantee the transferability of these parameters to different structural environments. The fitted parameters are then used to calculate the electronic structure of small-sized clusters of ZnS and CdS in given geometries and the results are compared with ab initio results. A fairly good agreement found in the one-electron energy spectrum and total energy confirms transferability of the parameters to different length scales. A detailed account of the calculation procedure and calibration results is given in the present paper. These parameters can be used to study the electronic structure of large-sized clusters where first-principles methods are computationally demanding. It may be mentioned that the SK parameters do not satisfy the R - (l + l' + 1) Harrison scaling law for larger values of the near-neighbor distance R.

  18. The cationic peptide LL-37 binds Mac-1 (CD11b/CD18) with a low dissociation rate and promotes phagocytosis.

    PubMed

    Zhang, Xianwei; Bajic, Goran; Andersen, Gregers R; Christiansen, Stig Hill; Vorup-Jensen, Thomas

    2016-05-01

    As a broad-spectrum anti-microbial peptide, LL-37 plays an important role in the innate immune system. A series of previous reports implicates LL-37 as an activator of various cell surface receptor-mediated functions, including chemotaxis in integrin CD11b/CD18 (Mac-1)-expressing cells. However, evidence is scarce concerning the direct binding of LL-37 to these receptors and investigations on the associated binding kinetics is lacking. Mac-1, a member of the β2 integrin family, is mainly expressed in myeloid leukocytes. Its critical functions include phagocytosis of complement-opsonized pathogens. Here, we report on interactions of LL-37 and its fragment FK-13 with the ligand-binding domain of Mac-1, the α-chain I domain. LL-37 bound the I-domain with an affinity comparable to the complement fragment C3d, one of the strongest known ligands for Mac-1. In cell adhesion assays both LL-37 and FK-13 supported binding by Mac-1 expressing cells, however, with LL-37-coupled surfaces supporting stronger cell adhesion than FK-13. Likewise, in phagocytosis assays with primary human monocytes both LL-37 and FK-13 enhanced uptake of particles coupled with these ligands but with a tendency towards a stronger uptake by LL-37.

  19. 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.

  20. 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

  1. Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region.

    PubMed

    Wooldridge, Linda; Lissina, Anna; Vernazza, Jonathan; Gostick, Emma; Laugel, Bruno; Hutchinson, Sarah L; Mirza, Fareed; Dunbar, P Rod; Boulter, Jonathan M; Glick, Meir; Cerundolo, Vincenzo; van den Berg, Hugo A; Price, David A; Sewell, Andrew K

    2007-05-01

    CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.

  2. 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.

  3. Colorectal Mucus Binds DC-SIGN and Inhibits HIV-1 Trans-Infection of CD4+ T-Lymphocytes

    PubMed Central

    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. PMID:25793526

  4. Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

    PubMed

    Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2007-11-01

    We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

  5. Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

    PubMed

    Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2007-11-01

    We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces. PMID:17947513

  6. CD and MCD Studies of the Effects of Component B Variant Binding on the Biferrous Active Site of Methane Monooxygenase†

    PubMed Central

    Mitić, Nataša; Schwartz, Jennifer K.; Brazeau, Brian J.; Lipscomb, John D.; Solomon, Edward I.

    2008-01-01

    The multi-component soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through activation of O2 at a non-heme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with Ligand Field calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O2 reactivity. MMOB variants that do not greatly increase O2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO. PMID:18627173

  7. Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual.

    PubMed

    Khan, Lubina; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Nair, Ambili; Andrabi, Raiees; Clark, Brenda E; Auyeung, Kate; Bhattacharya, Jayanta; Vajpayee, Madhu; Wig, Naveet; Pantophlet, Ralph; Luthra, Kalpana

    2015-01-01

    Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

  8. Role of membrane-anchored heparin-binding epidermal growth factor-like growth factor and CD9 on macrophages.

    PubMed Central

    Ouchi, N; Kihara, S; Yamashita, S; Higashiyama, S; Nakagawa, T; Shimomura, I; Funahashi, T; Kameda-Takemura, K; Kawata, S; Taniguchi, N; Matsuzawa, Y

    1997-01-01

    Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) is a potent mitogen for smooth-muscle cells (SMCs) belonging to the EGF family. We have previously determined that HB-EGF is expressed in macrophages and SMCs of human atherosclerotic lesions and that its membrane-anchored precursor, proHB-EGF, also has a juxtacrine mitogenic activity which is markedly enhanced by CD9, a surface marker of lymphohaemopoietic cells. Therefore, when both proHB-EGF and CD9 are expressed on macrophages, they may strongly promote the development of atherosclerosis. In the present study we have investigated the changes in proHB-EGF and CD9 in THP-1 cells during differentiation into macrophages and by the addition of oxidized low-density lipoproteins (OxLDL) and assessed juxtacrine growth activity of THP-1 macrophages for human aortic SMCs. HB-EGF and CD9 at both the mRNA and the protein level were up-regulated after differentiation into macrophages, and further expression of HB-EGF was induced by the addition of OxLDL or lysophosphatidylcholine. Juxtacrine induction by formalin-fixed growth was suppressed to control levels by an inhibitor of HB-EGF and was partially decreased by anti-CD9 antibodies. These results suggest that co-expression of proHB-EGF and CD9 on macrophages plays an important role in the development of atherosclerosis by a juxtacrine mechanism. PMID:9396739

  9. Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site.

    PubMed

    Subramanian, Shyamsundar; Boder, Eric T; Discher, Dennis E

    2007-01-19

    Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.

  10. RE-EXAMINATION OF CD91 FUNCTION IN GRP94 (gp96) SURFACE BINDING, UPTAKE AND PEPTIDE CROSS-PRESENTATION1

    PubMed Central

    Jockheck-Clark, Angela R.; Bowers, Edith V.; Totonchy, Mariam B.; Neubauer, Julie; Pizzo, Salvatore V.; Nicchitta, Christopher V.

    2013-01-01

    Extracellular GRP94 (gp96) can initiate both innate and adaptive immune responses through interactions with antigen presenting cell surface receptors. Following the identification of CD91 as a receptor functioning in the cross-presentation of GRP94-associated peptides, scavenger receptors SR-A and SREC-I were demonstrated to function in GRP94 cell surface binding and endocytosis, lending controversy to the assignment of CD91 as the unique GRP94 endocytic receptor. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in murine embyronic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNAi or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of the CD91 ligand receptor-associated protein (RAP); surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed following treatment of MEF cell lines with heparin, the sulfation inhibitor sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentaton of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to RAP or activated α2-macroglobulin and occurred primarily via a fluid phase uptake pathway. In summary, these data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis and peptide cross-presentation and identify HSPGs as novel GRP94 cell surface binding sites. PMID:21048103

  11. Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric.

    PubMed

    Lubelski, Jacek; van Merkerk, Ronald; Konings, Wil N; Driessen, Arnold J M

    2006-01-17

    LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs. PMID:16401093

  12. Isolation and characterization of a novel neutralizing antibody targeting the CD4-binding site of HIV-1 gp120.

    PubMed

    Qiao, Yuanyuan; Man, Lai; Qiu, Zonglin; Yang, Lingli; Sun, Youxiang; He, Yuxian

    2016-08-01

    Isolation and characterization of novel HIV-1 neutralizing antibodies assists the development of effective AIDS vaccines and immune therapeutics. In this study, we constructed a phage display antibody library by using the PBMC samples of a clade B' HIV-1-infected long-term nonprogressor (LTNP) whose sera exhibited broadly neutralizing activity. A novel human monoclonal antibody (hMAb), termed A16, was identified by panning the library with two clades of HIV-1 Env glycoproteins. We demonstrated that A16 neutralized 32% of 73 tested HIV-1 isolates and it targeted the CD4-binding site (CD4bs) of gp120 with high affinity. By selecting the peptide mimotopes in combination with computational algorithms and site-directed mutagenesis, the epitope of A16 was mapped to the structurally conserved sites located within the β1-α1, loop D, β20-β21 (bridging sheet) and β24-α5 of gp120, which critically determine the CD4 binding and are involved in the epitopes of CD4bs-directed antibodies. Our studies have shed new insights for the immune response of HIV-1 infection and offered a new tool for designing vaccine immunogens and antibody-based immune therapy. PMID:27387828

  13. Zn, Cu, Cd and Hg binding to metallothioneins in harbour porpoises Phocoena phocoena from the southern North Sea

    PubMed Central

    Das, Krishna; De Groof, Arnaud; Jauniaux, Thierry; Bouquegneau, Jean-Marie

    2006-01-01

    Background Harbour porpoises Phocoena phocoena from the southern North Sea are known to display high levels of Zn and Hg in their tissues linked to their nutritional status (emaciation). The question arises regarding a potential role of metallothioneins (MTs) with regard to these high metal levels. In the present study, metallothionein detection and associated Zn, Cd, Cu and Hg concentrations were investigated in the liver and kidney of 14 harbour porpoises collected along the Belgian coast. Results Metallothioneins seemed to play a key role in essential metal homeostasis, as they were shown to bind 50% of the total hepatic Zn and 36% of the total hepatic Cu concentrations. Renal MTs also participated in Cd detoxification, as they were shown to bind 56% of the total renal Cd. Hg was mainly found in the insoluble fraction of both liver and kidney. Concomitant increases in total Zn concentration and Zn bound to MTs were observed in the liver, whereas Zn concentration bound to high molecular weight proteins remained constant. Cu, Zn and Cd were accumulated preferentially in the MT fraction and their content in this fraction increased with the amount in the hepatocytosol. Conclusion MTs have a key role in Zn and Cu homeostasis in harbour porpoises. We demonstrated that increasing hepatic Zn concentration led to an increase in Zn linked to MTs, suggesting that these small proteins take over the Zn overload linked to the poor body condition of debilitated harbour porpoises. PMID:16464247

  14. 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.

  15. Phage randomization in a charybdotoxin scaffold leads to CD4-mimetic recognition motifs that bind HIV-1 envelope through non-aromatic sequences.

    PubMed

    Li, C; Dowd, C S; Zhang, W; Chaiken, I M

    2001-06-01

    Binding of HIV-1 gp120 to T-cell receptor CD4 initiates conformational changes in the viral envelope that trigger viral entry into host cells. Phage epitope randomization of a beta-turn loop of a charybdotoxin-based miniprotein scaffold was used to identify peptides that can bind gp120 and block the gp120-CD4 interaction. We describe here the display of the charybdotoxin scaffold on the filamentous phage fUSE5, its use to construct a beta-turn library, and miniprotein sequences identified through library panning with immobilized Env gp120. Competition enzyme-linked immunosorbent assay (ELISA) identified high-frequency phage selectants for which specific gp120 binding was competed by sCD4. Several of these selectants contain hydrophobic residues in place of the Phe that occurs in the gp120-binding beta-turns of both CD4 and previously identified scorpion toxin CD4 mimetics. One of these selectants, denoted TXM[24GQTL27], contains GQTL in place of the CD4 beta-turn sequence 40QGSF43. TXM[24GQTL27] peptide was prepared using solid-phase chemical synthesis, its binding to gp120 demonstrated by optical biosensor kinetics analysis and its affinity for the CD4 binding site of gp120 confirmed by competition ELISA. The results demonstrate that aromatic-less loop-containing CD4 recognition mimetics can be formed with detectable envelope protein binding within a beta-turn of the charybdotoxin miniprotein scaffold. The results of this work establish a methodology for phage display of a charybdotoxin miniprotein scaffold and point to the potential value of phage-based epitope randomization of this miniprotein for identifying novel CD4 mimetics. The latter are potentially useful in deconvoluting structural determinants of CD4-HIV envelope recognition and possibly in designing antagonists of viral entry. PMID:11437954

  16. 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.

  17. 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

    2015-09-01

    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.

  18. 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.

  19. 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.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; et al

    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

  20. Strain-specific V3 and CD4 binding site autologous HIV-1 neutralizing antibodies select neutralization-resistant viruses

    PubMed Central

    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; Shaw, George M.; Sodroski, Joseph G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-01-01

    Summary 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 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. PMID:26355218

  1. Lipopolysaccharide Binding Protein and sCD14 are Not Produced as Acute Phase Proteins in Cardiac Surgery

    PubMed Central

    Kudlova, Manuela; Kunes, Pavel; Kolackova, Martina; Lonsky, Vladimir; Mandak, Jiri; Andrys, Ctirad; Jankovicova, Karolina; Krejsek, Jan

    2007-01-01

    Objectives. The changes in the serum levels of lipopolysaccharide binding protein (LBP) and sCD14 during cardiac surgery were followed in this study. Design. Thirty-four patients, 17 in each group, were randomly assigned to coronary artery bypass grafting surgery performed either with (“on-pump”) or without (“off-pump”) cardiopulmonary bypass. LBP and sCD14 were evaluated by ELISA. Results. The serum levels of LBP were gradually increased from the 1st postoperative day and reached their maximum on the 3rd postoperative day in both “on-pump” and “off-pump” patients (30.33±9.96 μg/mL; 37.99±16.58 μg/mL), respectively. There were no significant differences between “on-pump” and “off-pump” patients regarding LBP. The significantly increased levels of sCD14 from the 1st up to the 7th postoperative day in both “on-pump” and “off-pump” patients were found with no significant differences between these groups. No correlations between LBP and sCD14 and IL-6, CRP and long pentraxin PTX3 levels were found. Conclusions. The levels of LBP and sCD14 are elevated in cardiac surgical patients being similar in both groups. These molecules are not produced as acute phase proteins in these patients. PMID:18288274

  2. Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

    PubMed Central

    Wyatt, R; Thali, M; Tilley, S; Pinter, A; Posner, M; Ho, D; Robinson, J; Sodroski, J

    1992-01-01

    Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions. Images PMID:1279195

  3. A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

    PubMed Central

    Freund, Natalia T.; Horwitz, Joshua A.; Nogueira, Lilian; Sievers, Stuart A.; Scharf, Louise; Scheid, Johannes F.; Gazumyan, Anna; Liu, Cassie; Velinzon, Klara; Goldenthal, Ariel; Sanders, Rogier W.; Moore, John P.; Bjorkman, Pamela J.; Seaman, Michael S.; Walker, Bruce D.; Klein, Florian; Nussenzweig, Michel C.

    2015-01-01

    The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans. PMID:26516768

  4. Keratinocytes-associated chemokines and enzymatically quiescent heparanase induce the binding of resting CD4+ T cells.

    PubMed

    Hershkoviz, R; Marikovsky, M; Gilat, D; Lider, O

    1996-02-01

    Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli. PMID:8601723

  5. 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

  6. 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.

  7. Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans

    PubMed Central

    Mir, Gerard Hernandez; Helin, Jari; Skarp, Kari-Pekka; Cummings, Richard D.; Mäkitie, Antti; Renkonen, Risto

    2009-01-01

    Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLex). It is thought that multivalent 6-sulfo SLex expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLex in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin–binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLex epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLex. N-glycans containing potential 6-sulfo SLex epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLex epitopes on O-glycans that are important for its recognition by L-selectin. PMID:19359410

  8. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    PubMed

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

  9. Induction of tumor necrosis factor production from monocytes stimulated with mannuronic acid polymers and involvement of lipopolysaccharide-binding protein, CD14, and bactericidal/permeability-increasing factor.

    PubMed Central

    Jahr, T G; Ryan, L; Sundan, A; Lichenstein, H S; Skjåk-Braek, G; Espevik, T

    1997-01-01

    Well-defined polysaccharides, such as beta1-4-linked D-mannuronic acid (poly[M]) derived from Pseudomonas aeruginosa, induce monocytes to produce tumor necrosis factor (TNF) through a pathway involving membrane CD14. In this study we have investigated the effects of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing factor (BPI) on poly(M) binding to monocytes and induction of TNF production. We show that LBP increased the TNF production from monocytes stimulated with poly(M). Addition of sCD14 alone had only minor effects, but when it was added together with LBP, a rise in TNF production was seen. BPI was found to inhibit TNF production from monocytes stimulated with poly(M) in the presence of LBP, LBP-sCD14, or 10% human serum. Binding studies showed that poly(M) bound to LBP- and BPI-coated immunowells, while no significant binding of poly(M) to sCD14-coated wells in the absence of serum was observed. Binding of poly(M) to monocytes was also examined by flow cytometry, and it was shown that the addition of LBP or 10% human serum clearly increased the binding of poly(M) to monocytes. BPI inhibited the binding of poly(M) to monocytes in the presence of LBP, LBP-sCD14, or 10% human serum. Our data demonstrate a role for LBP, LBP-sCD14, and BPI in modulating TNF responses of defined polysaccharides. PMID:8975896

  10. Binding of Full-Length HIV-1 gp120 to CD4 Induces Structural Reorientation around the gp120 Core

    SciTech Connect

    Ashish,F.; Garg, R.; Anguita, J.; Krueger, J.

    2006-01-01

    Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.

  11. 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

  12. Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).

    PubMed Central

    Bergelson, J M; Mohanty, J G; Crowell, R L; St John, N F; Lublin, D M; Finberg, R W

    1995-01-01

    A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment. PMID:7531780

  13. MHC class I molecules with Superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs.

    PubMed

    Wooldridge, Linda; Clement, Mathew; Lissina, Anna; Edwards, Emily S J; Ladell, Kristin; Ekeruche, Julia; Hewitt, Rachel E; Laugel, Bruno; Gostick, Emma; Cole, David K; Debets, Reno; Berrevoets, Cor; Miles, John J; Burrows, Scott R; Price, David A; Sewell, Andrew K

    2010-04-01

    CD8(+) CTLs are essential for effective immune defense against intracellular microbes and neoplasia. CTLs recognize short peptide fragments presented in association with MHC class I (MHCI) molecules on the surface of infected or dysregulated cells. Ag recognition involves the binding of both TCR and CD8 coreceptor to a single ligand (peptide MHCI [pMHCI]). The TCR/pMHCI interaction confers Ag specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to Ag. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. In this study, we show that increasing the strength of the pMHCI/CD8 interaction by approximately 15-fold results in nonspecific, cognate Ag-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with superenhanced affinity for CD8 activate CTLs in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL Ag specificity.

  14. Surface complexation of aluminum on isolated fish gill cells

    SciTech Connect

    Wilkinson, K.J. Campbell, G.C.; Bertsch, P.M.; Jagoe, C.H.

    1993-06-01

    Cells from the gills of largemouth bass (Micropterus salmoides) were isolated and exposed to dilute solutions of Al, Al in the presence of fluoride, or Al plus dissolved organic matter (DOM) to determine the cells` metal binding potential in an acidic medium. Microelectrophoresis was employed to monitor the extent of aluminum sorption to cells in the presence of added ligand. In the absence of Al, the gill cells exhibit an appreciable negative charge; Al binding to the cell surface increases the electric potential at the shear plane and leads to a reduction in the cell`s (negative) electrophoretic mobility. In the presence of both Al and F, aluminum complexation at the gill surface is only marginally reduced; the formation of a mixed ligand complex, [F-Al-L-cell], is proposed to account for the observed results. The presence of such ternary complexes was subsequently verified by {sup 19}F nuclear magnetic resonance spectroscopy and by potentiometry. Addition of DOM increased the negative electrophoretic mobility of the isolated gill cells both in the presence and absence of aluminum (7.4 {mu}M). 45 refs., 7 figs., 1 tab.

  15. Single-molecule binding of CD44 to fibrin versus P-selectin predicts their distinct shear-dependent interactions in cancer

    PubMed Central

    Raman, Phrabha S.; Alves, Christina S.; Wirtz, Denis; Konstantopoulos, Konstantinos

    2011-01-01

    P-selectin and fibrin(ogen) have pivotal roles in the hematogenous dissemination of tumor cells. CD44 variant isoforms, CD44v, have been identified as the major functional P-selectin ligands and fibrin receptors on metastatic colon carcinoma cells. The molecular recognition of CD44v by fibrin mediates firm adhesion at low shear, whereas CD44v–P-selectin binding supports transient rolling interactions at elevated shear stresses and low site densities of P-selectin. We used single-molecule force spectroscopy to provide a molecular interpretation for these two distinct adhesion events. The CD44v–P-selectin bond has a longer unstressed equilibrium lifetime, a lower reactive compliance and a higher tensile strength relative to the CD44v–fibrin bond. These intrinsic differences confer the ability to the CD44v–P-selectin pair to mediate binding at higher shear stresses. Increasing the duration of receptor–ligand contact (2–200 milliseconds) did not affect the micromechanical properties of the CD44v–P-selectin bond, but it increased the tensile strength and the depth of the free energy barrier of the CD44v–fibrin bond and decreased its reactive compliance. This bond strengthening at longer interaction times might explain why CD44v binding to immobilized fibrin occurs at low shear. Single-molecule characterization of receptor–ligand binding can predict the shear-dependent adhesive interactions between cells and substrates observed both in vitro and in vivo. PMID:21558419

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

    PubMed Central

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

    2012-01-01

    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 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. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells. PMID:22841832

  17. Cadmium inhibition of Ca sup 2+ uptake in rainbow trout gills

    SciTech Connect

    Verbost, P.M.; Flik, G.; Lock, R.A.C.; Wendelaar Bonga, E.E. )

    1987-08-01

    The effects of cadmium (Cd{sup 2+}) on calcium (Ca{sup 2+}) transport in the gills of rainbow trout (Salmo gairdneri) were studied. The gill epithelium of freshwater fish represents a model for a Ca{sup 2+}-transporting tight epithelium. Unidirectional {sup 45}Ca{sup 2+} fluxes in the gills were estimated in an isolated saline-perfused heat preparation. {sup 45}Ca{sup 2+} influx was not affected when up to 10 {mu}M Cd were added to the ventilatory water at the start of flux determinations (in vitro exposure). However, after 16 h in vivo preexposure of the fish to 0.1 {mu}M Cd in the water, a 79% inhibition of Ca{sup 2+} influx was observed. Ca{sup 2+} efflux was not affected when up to 10 {mu}M Cd were added to the ventilatory water during the flux determination. Ca{sup 2+} efflux in fish preexposed to 0.1 {mu}M Cd for 16 h was also not affected; a preexposure to 1 {mu}M Cd, however, resulted in a 173% increase in Ca{sup 2+} efflux rates. Tracer retention in the gill tissue indicated that both Ca{sup 2+} and Cd{sup 2+} enter the gill epithelium via a lanthanum (La{sup 3+})-inhibitable pathway. It is concluded that Cd{sup 2+} readily enters the branchial epithelial cells, similarly as Ca{sup 2+} does via La{sup 3+}-sensitive apical Ca{sup 2+} channels. The inhibitory action of Cd{sup 2+} on transepithelial Ca{sup 2+} influx seems to result from an inhibition of the basolateral Ca{sup 2+} transport, occurring after a critical intracellular Cd{sup 2+} concentration has been reached.

  18. Thrombin-cleaved COOH(-) terminal osteopontin peptide binds with cyclophilin C to CD147 in murine breast cancer cells.

    PubMed

    Mi, Zhiyong; Oliver, Tim; Guo, Hongtao; Gao, Chengjiang; Kuo, Paul C

    2007-05-01

    Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombin-cleaved osteopontin COOH-terminal fragment.

  19. Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26

    SciTech Connect

    Gonzalez-Gronow, Mario . E-mail: gonza002@mc.duke.edu; Grenett, Hernan E.; Gawdi, Govind; Pizzo, Salvatore V.

    2005-02-01

    Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2{epsilon}, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2{epsilon} to CD26 on the cell surface induces a Ca{sup 2+} signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel registered . We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2{epsilon} binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2{epsilon}. As a result, the plasminogen 2{epsilon}-induced Ca{sup 2+} signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel registered by 1-LN cells is blocked. Angiostatin 2{epsilon} is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2{epsilon} may also directly block tumor metastasis.

  20. Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release.

    PubMed

    Soell, M; Lett, E; Holveck, F; Schöller, M; Wachsmann, D; Klein, J P

    1995-01-15

    The present work was initiated to define mechanisms that account for the binding on human monocytes of streptococcal cell wall polysaccharides formed by rhamnose glucose polymers (RGPs), and subsequent stimulatory activities. We show here that RGPs bind to and stimulate human monocytes to produce TNF-alpha in a dose-dependent manner. To detect cell surface RGPs binding proteins, intact monocytes were biotinylated before lysis with Nonidet P-40 and solubilized proteins were incubated with RGPs Affi-Prep beads. One major membrane protein of 55 kDa was specifically detected and identified as CD14 because it reacted with anti-CD14 mAbs. Furthermore, anti-CD14 mAbs were able to perform a dose-dependent inhibition of RGPs binding, and suppressed TNF-alpha release from RGPs-stimulated monocytes. Moreover, we demonstrated that RGPs also bind to CD11b; however, this binding is not implicated in synthesis of TNF-alpha. Interestingly, RGPs binding to monocytes was enhanced by human normal serum (HNS) whereas HNS inhibits the TNF-alpha-stimulating activity of RGPs. Western blotting analysis of HNS proteins purified on RGPs Affi-prep beads revealed three specific bands of 75, 55, and 32 kDa reactive with anti-C3 Abs, anti-CD14 mAbs (TUK4), and anti-human mannan binding protein (hMBP)-derived peptide IgG, respectively. These results suggest that C3, soluble CD14, and hMBP form complexes that are probably active in enhancing the binding of RGPs to monocytes. Additional studies have shown that hMBP that recognizes RGPs prevents, unlike the LPS binding protein, TNF-alpha release by inhibiting the binding of RGPs to CD14 Ag. By incubating cells with a constant amount of RGPs-hMBP complexes in the presence or absence of increasing concentrations of C1q, we also demonstrated that C1q receptor mediates the binding and probably the uptake of RGPs-hMBP complexes by human monocytes. PMID:7529289

  1. Lubricin/Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of Lubricin’s suppression of Pro-inflammatory Cytokine Induced Synoviocyte Proliferation

    PubMed Central

    Al-Sharif, Afnan; Jamal, Maha; Zhang, Ling; Larson, Katherine; Schmidt, Tannin; Jay, Gregory; Elsaid, Khaled

    2015-01-01

    Objective To evaluate recombinant human proteoglycan 4 (rhPRG4) binding to CD44 receptor and its consequence on cytokine induced synoviocyte proliferation. Methods rhPRG4 binding to CD44 and competition with high molecular weight hyaluronic acid (HMW HA) was evaluated using a direct enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance. Sialidase-A and O-glycosidase digestion of rhPRG4 was performed and CD44 binding was evaluated using ELISA. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were stimulated with interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) for 48 hours in the presence or absence of rhPRG4 or HMW HA at 20, 40 and 80μg/ml and cell proliferation was measured. CD44 contribution was assessed by co-incubation with a CD44 antibody (IM7). The anti-proliferative effect of rhPRG4 was investigated following treatment of Prg4−/− synoviocytes with IL-1β or TNF-α in the presence or absence of IM7. Results rhPRG4 binds CD44 and interferes with HMW HA CD44 binding. Removal of sialic acid and O-glycosylations significantly increased CD44 binding by rhPRG4 (p<0.001). rhPRG4 and HMW HA at 40 and 80μg/ml significantly suppressed IL-1β induced RA-FLS proliferation (p<0.05). rhPRG4 at 20, 40 and 80μg/ml significantly suppressed TNF-α induced RA-FLS proliferation (p<0.05). CD44 neutralization reversed the effect of rhPRG4 on IL-1β and TNF-α stimulated RA-FLS and the effect of HMW HA on IL-1β stimulated RA-FLS. rhPRG4 inhibited cytokine-induced proliferation of Prg4−/− synoviocytes which could be prevented by blocking CD44. Conclusion Lubricin is a novel putative ligand for CD44 and may control synoviocyte overgrowth in inflammatory arthropathies via a CD44-mediated mechanism. PMID:25708025

  2. 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.

  3. 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.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; et al

    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

  4. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding

    PubMed Central

    Lin, Pin-Fang; Blair, Wade; Wang, Tao; Spicer, Timothy; Guo, Qi; Zhou, Nannan; Gong, Yi-Fei; Wang, H.-G. Heidi; Rose, Ronald; Yamanaka, Gregory; Robinson, Brett; Li, Chang-Ben; Fridell, Robert; Deminie, Carol; Demers, Gwendeline; Yang, Zheng; Zadjura, Lisa; Meanwell, Nicholas; Colonno, Richard

    2003-01-01

    BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 μM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 μM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs. PMID:12930892

  5. 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

  6. 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. PMID:25668665

  7. 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.

  8. 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. PMID:8562500

  9. Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners

    PubMed Central

    Muramatsu, Takashi

    2016-01-01

    Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. PMID:26684586

  10. 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.

  11. 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.

  12. An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

    NASA Astrophysics Data System (ADS)

    Gupta, Sebanti; Chit, Joel Chia-Yeong; Feng, Chen; Bhunia, Anirban; Tan, Suet-Mien; Bhattacharjya, Surajit

    2015-06-01

    Integrins are involved in cell migration and adhesion. A large number of proteins interact with the cytoplasmic tails of integrins. Dok1 is a negative regulator of integrin activation and it binds to the phosphorylated membrane proximal NxxY motif in a number of integrin β tails. The β tail of the β2 integrins contains a non-phosphorylatable NxxF motif. Hence it is unclear how Dok1 associates with the β2 integrins. We showed in this study using NMR and cell based analyses that residues Ser745 and Ser756 in the integrin β2 tail, which are adjacent to the NxxF motif, are required for Dok1 interaction. NMR analyses detected significant chemical shift changes and higher affinity interactions between Dok1 phospho-tyrosine binding (PTB) domain and integrin β2 tail peptide containing pSer756 compared to pSer745. The phosphorylated β2 peptide occupies the canonical ligand binding pocket of Dok1 based on the docked structure of the β2 tail-Dok1 PTB complex. Taken together, our data suggest an alternate phosphorylation switch in β2 integrins that regulates Dok1 binding. This could be important for cells of the immune system and their functions.

  13. An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding.

    PubMed

    Gupta, Sebanti; Chit, Joel Chia-Yeong; Feng, Chen; Bhunia, Anirban; Tan, Suet-Mien; Bhattacharjya, Surajit

    2015-06-25

    Integrins are involved in cell migration and adhesion. A large number of proteins interact with the cytoplasmic tails of integrins. Dok1 is a negative regulator of integrin activation and it binds to the phosphorylated membrane proximal NxxY motif in a number of integrin β tails. The β tail of the β2 integrins contains a non-phosphorylatable NxxF motif. Hence it is unclear how Dok1 associates with the β2 integrins. We showed in this study using NMR and cell based analyses that residues Ser745 and Ser756 in the integrin β2 tail, which are adjacent to the NxxF motif, are required for Dok1 interaction. NMR analyses detected significant chemical shift changes and higher affinity interactions between Dok1 phospho-tyrosine binding (PTB) domain and integrin β2 tail peptide containing pSer756 compared to pSer745. The phosphorylated β2 peptide occupies the canonical ligand binding pocket of Dok1 based on the docked structure of the β2 tail-Dok1 PTB complex. Taken together, our data suggest an alternate phosphorylation switch in β2 integrins that regulates Dok1 binding. This could be important for cells of the immune system and their functions.

  14. 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).

  15. 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).

  16. Dextran Sulfate Suppression of Viruses in the HIV Family: Inhibition of Virion Binding to CD4+ Cells

    NASA Astrophysics Data System (ADS)

    Mitsuya, Hiroaki; Looney, David J.; Kuno, Sachiko; Ueno, Ryuji; Wong-Staal, Flossie; Broder, Samuel

    1988-04-01

    The first step in the infection of human T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is attachment to the target cell receptor, the CD4 antigen. This step may be vulnerable to attack by antibodies, chemicals, or small peptides. Dextran sulfate (molecular weight approximately 8000), which has been given to patients as an anticoagulant or antilipemic agent for more than two decades, was found to block the binding of virions to various target T lymphocytes, inhibit syncytia formation, and exert a potent inhibitory effect against HIV-1 in vitro at concentrations that may be clinically attainable in human beings. This drug also suppressed the replication of HIV-2 in vitro. These observations could have theoretical and clinical implications in the strategy to develop drugs against HIV types 1 and 2.

  17. Design, synthesis and biological evaluation of small molecule inhibitors of CD4-gp120 binding based on virtual screening

    PubMed Central

    Elban, Mark A.; Courter, Joel R.; Sugawara, Akihiro; Soeta, Takahiro; Madani, Navid; Princiotto, Amy M.; Kwon, Young Do; Kwong, Peter D.; Schön, Arne; Freire, Ernesto; Sodroski, Joseph; Smith, Amos B.

    2011-01-01

    The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, GOLD docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogues that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogues of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogues provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein-ligand interactions in the vestibule of gp120 Phe 43 cavity. PMID:21169023

  18. Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro.

    PubMed Central

    Ockenhouse, C F; Magowan, C; Chulay, J D

    1989-01-01

    The CD36 leukocyte differentiation antigen, recognized by MAbs OKM5 and OKM8 and found on human monocytes and endothelial cells, has been implicated as a sequestration receptor for erythrocytes infected with the human malaria parasite Plasmodium falciparum (IRBC). CD36 is also expressed on platelets and appears to be identical to platelet glycoprotein IV. We investigated receptor activation of monocytes and platelets by anti-CD36 MAbs and by IRBC. Incubation of human monocytes with anti-CD36 MAbs or IRBC resulted in stimulation of the respiratory burst as measured by reduction of nitroblue tetrazolium and generation of chemiluminescence. Incubation of human platelets with anti-CD36 MAbs resulted in platelet activation as measured by aggregation or ATP secretion. Activation of monocytes and platelets required appropriate intracellular transmembrane signaling and was inhibited by calcium antagonists or by specific inhibitors of protein kinase C or guanine nucleotide binding proteins. Soluble CD36 inhibited binding of IRBC to both monocytes and platelets, suggesting that these interactions are mediated by the CD36 receptor. Using a cytochemical electron microscopic technique, the presence of reactive oxygen intermediates was identified at the interface between human monocytes and IRBC. These data provide support for the hypothesis that reactive oxygen intermediates produced by monocytes when IRBC ligands interact with cell surface receptors may play a role in the pathophysiology of falciparum malaria. Images PMID:2474569

  19. Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock.

    PubMed

    Voss, Oliver H; Murakami, Yousuke; Pena, Mirna Y; Lee, Ha-Na; Tian, Linjie; Margulies, David H; Street, Jonathan M; Yuen, Peter S T; Qi, Chen-Feng; Krzewski, Konrad; Coligan, John E

    2016-06-21

    Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS. PMID:27261276

  20. CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines.

    PubMed

    Kijimoto-Ochiai, S; Uede, T

    1995-06-01

    Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Effect of CD44 Binding Peptide Conjugated to an Engineered Inert Matrix on Maintenance of Breast Cancer Stem Cells and Tumorsphere Formation

    PubMed Central

    Moeinzadeh, Seyedsina; He, Xuezhong; Jabbari, Esmaiel

    2013-01-01

    Introduction As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs. Methods 4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). Results Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. Conclusion CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors. PMID:23527117

  7. Thermodynamics of Cd2+ and Zn2+ binding by the phytochelatin (gamma-Glu-Cys)4-Gly and its precursor glutathione.

    PubMed

    Chekmeneva, Elena; Prohens, Rafel; Díaz-Cruz, José Manuel; Ariño, Cristina; Esteban, Miquel

    2008-04-01

    Isothermal titration calorimetry (ITC) was used to study the binding of Cd(2+) and Zn(2+) by glutathione (GSH) and phytochelatins (PC(n)), the metal sequestering compounds in plants and algae. The results are compared with those obtained by differential pulse polarography (DPP) assisted by multivariate curve resolution with alternating least squares (MCR-ALS) and by electrospray ionization mass spectrometry (ESI-MS). ITC allows one to determine (i) the stoichiometries of the different complexes (and confirms those found by DPP/MCR-ALS and ESI-MS) and (ii) their binding and thermodynamic parameters. For Cd-PC(4), the sequential binding sites model with two identical sites yields the best fitting of ITC curves and confirms the presence of CdPC(4) and Cd(2)PC(4) complexes. For Zn-PC(4), exothermic formation of ZnPC(4) is reported. Conditional stability and formation constants for Cd-GSH and Zn-GSH are determined from the fitting of the proper model to experimental ITC curves. The effect of different buffers in the complexation processes shows the key role of the choice of the buffer in calorimetric study.

  8. Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites.

    PubMed

    Leone, Francisco Assis; Masui, Douglas Chodi; de Souza Bezerra, Thais Milena; Garçon, Daniela Pereira; Valenti, Wagner Cotroni; Augusto, Alessandra Silva; McNamara, John Campbell

    2012-04-01

    We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

  9. 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.

  10. 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.

  11. 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.

  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. Histological changes in the kidneys and gills of the stickleback, Gasterosteus aculeatus L, exposed to dissolved cadmium in hard water

    SciTech Connect

    Oronsaye, J.A.

    1989-06-01

    Damage to the kidneys and gills of the three spined stickleback Gasterosteus aculeatus have been investigated, using light microscopy after exposure of fish to 2 to 6 mg Cd2+ liter-1 hard water (299 mg liter-1 as CaCO/sub 3/). Cytological breakdown of kidneys and gill tissue appears to be the cause of death, since oxygen uptake and cadmium excretion are impaired, leading to tissue hypoxia and fish poisoning.

  14. Functional epitope analysis of the human CD4 molecule: antibodies that inhibit human immunodeficiency virus type 1 gene expression bind to the immunoglobulin CDR3-like region of CD4.

    PubMed Central

    Benkirane, M; Hirn, M; Carrière, D; Devaux, C

    1995-01-01

    We recently demonstrated that monoclonal antibody (MAb) 13B8-2, specific for the immunoglobulin (Ig) complementary determining region 3 (CDR3)-like region of the CD4 molecule, inhibits viral transcription in human immunodeficiency virus (HIV)-infected CEM cells and HIV type 1 (HIV-1) promoter activity. Here, we have studied the capacity of several MAb specific for the D1 domain of CD4, including anti-CDR2-like (Leu-3a and ST4) and anti-CDR3-like (13B8-2 and ST40) MAb, and for the D2 domain of CD4 (BL4) to inhibit both provirus transcription in HIV-1LAI-infected CEM cells and transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the HIV-1 long terminal repeat in transiently transfected CEM cells. We found that HIV-1 promoter activity and provirus transcription are inhibited only by MAb that bind to the CDR3-like region in domain 1 of CD4. Moreover, we demonstrated that the Fab fragment of an anti-CDR3-like region-specific anti-CD4 MAb is a powerful inhibitor of HIV-1 promoter activity. These results have implications for understanding the role of the CDR3-like region in CD4 T-cell signaling, which controls provirus transcription. PMID:7474106

  15. Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+ and CD8+ T Cells Leads to Impaired Activation of Lck and Akt

    PubMed Central

    Yao, Zhi Qiang; Eisen-Vandervelde, Audrey; Waggoner, Stephen N.; Cale, Evan M.; Hahn, Young S.

    2004-01-01

    Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 × 10−7 M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8+ than on CD4+ T cells, resulting in more severe core-induced suppression of the CD8+-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation. PMID:15163734

  16. 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

  17. Endothelial-binding, Proinflammatory T Cells Identified by MCAM (CD146) Expression: Characterization and Role in Human Autoimmune Diseases

    PubMed Central

    Dagur, Pradeep K; McCoy, J Philip

    2015-01-01

    A subset of T cells defined by the cell surface expression of MCAM (CD146) has been identified in the peripheral circulation of healthy individuals. These cells comprise approximately 3% of the pool of circulating T cells, have an effector memory phenotype, and are capable of producing several cytokines. Notably, the MCAM positive cells are enhanced for IL-17 production compared to MCAM negative effector memory T cells. These call are committed for IL-17 production and do not require in vitro polarization with exogenous cytokines. MCAM positive T cells also demonstrate an increased ability to bind to endothelial monolayers. In numerous autoimmune diseases these cells are found at increased proportions in the peripheral circulation, and at the sites of active inflammation in patients with autoimmune disease, these cells appear in large numbers are major contributors to IL-17 production. Studies to date have been performed with human subjects and it is uncertain if appropriate mouse models exist for this cells type. These cells could represent early components of the adaptive immune response and serve as targets of therapy in these diseases, although much work remains to be performed in order to discern the exact nature and function of these cells. PMID:25595133

  18. 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

  19. EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

    PubMed Central

    Mohan Kumar, Dipu; Lin, Mingqun; Xiong, Qingming; Webber, Mathew James; Kural, Comert

    2015-01-01

    ABSTRACT Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. PMID:26530384

  20. Identification of the Cu2+ Binding Sites in the N-Terminal Domain of the Prion Protein by EPR and CD Spectroscopy†

    PubMed Central

    Aronoff-Spencer, Eliah; Burns, Colin S.; Avdievich, Nikolai I.; Gerfen, Gary J.; Peisach, Jack; Antholine, William E.; Ball, Haydn L.; Cohen, Fred E.; Prusiner, Stanley B.; Millhauser, Glenn L.

    2010-01-01

    Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60–91. This region selectively binds divalent copper ions (Cu2+) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu2+–peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu2+ binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu2+/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu2+ is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu2+ is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides. PMID:11076515

  1. 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. PMID:20965062

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

    PubMed Central

    FISCHER, Mark W. F.; MONEY, Nicholas P.

    2009-01-01

    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 freestanding 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. PMID:20965062

  3. High-resolution crystal structures of alternate forms of the human CD44 hyaluronan-binding domain reveal a site for protein interaction

    PubMed Central

    Liu, Li-Kai; Finzel, Barry

    2014-01-01

    Two new crystal structures of the extracellular hyaluronan-binding domain of human CD44 are described at high resolution. A hexagonal crystal form at 1.60 Å resolution and a monoclinic form at 1.08 Å resolution both have two molecules in the asymmetric unit arranged about a similar noncrystallographic twofold axis of symmetry. These structures are compared with those previously reported at 2.20 Å resolution to show that the fold is quite resistant to structural deformation in different crystal environments. Unexpectedly, a short peptide is found in the monoclinic crystals at a site remote from the known hyaluronan-binding groove. The peptide with a valine at the carboxy-terminus must have co-purified from the bacterial expression host and binds on the opposite side of the domain from the known hyaluronan-binding groove. This opportunistic binding may identify a site of interaction used as CD44 assembles with other proteins to accomplish effective signaling regarding changes to the extracellular environment. PMID:25195884

  4. Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

    PubMed Central

    Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

    2001-01-01

    The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

  5. Polymorphisms at the microRNA binding-site of the stem cell marker gene CD133 modify susceptibility to and survival of gastric cancer.

    PubMed

    Wang, Qiming; Liu, Hongliang; Xiong, Huihua; Liu, Zhensheng; Wang, Li-E; Qian, Ji; Muddasani, Ramya; Lu, Victoria; Tan, Dongfeng; Ajani, Jaffer A; Wei, Qingyi

    2015-06-01

    CD133 is one of the most common stem cell markers, and functional single nucleotide polymorphisms (SNPs) of CD133 may modulate its gene functions and thus cancer risk and patient survival. We hypothesized that potentially functional CD133 SNPs are associated with gastric cancer (GC) risk and survival. To test this hypothesis, we conducted a case-control study of 371 GC patients and 313 cancer-free controls frequency-matched by age, sex, and ethnicity. We genotyped four selected, potentially functional CD133 SNPs (rs2240688A>C, rs7686732C>G, rs10022537T>A, and rs3130C>T) and used logistic regression analysis for associations of these SNPs with GC risk and Cox hazards regression analysis for survival. We found that compared with the miRNA binding site rs2240688 AA genotype, AC + CC genotypes were associated with significantly increased GC risk (adjusted OR = 1.52, 95% CI = 1.09-2.13); for another miRNA binding site rs3130C>T SNP, the TT genotype was associated with significantly reduced GC risk (adjusted OR = 0.68, 95% CI = 0.48-0.97), compared with CC + CT genotypes. In all patients, the risk rs3130 TT variant genotype was significantly associated with overall survival (OS) (adjusted P(trend) = 0.016 and 0.007 under additive and recessive models, respectively). These findings suggest that these two CD133 miRNA binding site variants, rs2240688 and rs3130, may be potential biomarkers for genetic susceptibility to GC and possible predictors for survival in GC patients but require further validation by larger studies.

  6. Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation

    PubMed Central

    Matsukawa, Toshihiro; Izawa, Kumi; Isobe, Masamichi; Takahashi, Mariko; Maehara, Akie; Yamanishi, Yoshinori; Kaitani, Ayako; Okumura, Ko; Teshima, Takanori; Kitamura, Toshio; Kitaura, Jiro

    2016-01-01

    Objective Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide–LMIR3 interaction in the development of IBD. Design The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3−/−, mast cell-deficient KitW-sh/W-sh, KitW-sh/W-shLMIR3−/− or KitW-sh/W-sh mice engrafted with WT or LMIR3−/− bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. Results LMIR3 deficiency exacerbated DSS-induced colitis in mice. KitW-sh/W-sh mice harbouring LMIR3−/− mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide–LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide–LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. Conclusions LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3−/− mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide–LMIR3 binding. PMID:25673319

  7. The counterreceptor binding site of human CD2 exhibits an extended surface patch with multiple conformations fluctuating with millisecond to microsecond motions.

    PubMed Central

    Wyss, D. F.; Dayie, K. T.; Wagner, G.

    1997-01-01

    We have used 15N NMR relaxation experiments to probe, for the glycosylated human CD2 adhesion domain, the overall molecular motion, as well as very fast nanosecond-picosecond (ns-ps) and slow millisecond-microsecond (ms-microsecond) internal motions. Using a novel analysis method that considers all residues, we obtained a correlation time for the overall motion of 9.5 +/- 0.3 ns. Surprisingly, we found a large contiguous patch of residues in the counterreceptor (CD58) binding site of human CD2 exhibiting slow conformational exchange motions (ms-microsecond). On the other hand, almost none of the residues of the CD58 binding side display fast (ns-ps) internal motions of amplitudes larger than what is seen for well-ordered regions of the structure. Residues close to the N-glycosylation site, and the first N-acetylglucosamine of the high mannose glycan are as rigid as the protein core. Residues conserved in the immunoglobulin superfamily V-set domain are generally very rigid. PMID:9070436

  8. Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness*

    PubMed Central

    Irving, Melita; Zoete, Vincent; Hebeisen, Michael; Schmid, Daphné; Baumgartner, Petra; Guillaume, Philippe; Romero, Pedro; Speiser, Daniel; Luescher, Immanuel; Rufer, Nathalie; Michielin, Olivier

    2012-01-01

    Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1157–165-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8+ T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8+ T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca2+ flux for CD8+ T cells expressing TCR within a dissociation constant (KD) range of ∼1–5 μm. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with KD < ∼1 μm, irrespective of CD8 co-engagement and of half-life (t1/2 = ln 2/koff) values. With increased peptide concentration, however, the activity levels of CD8+ T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8+ T cells, as well as for peptide vaccination strategies. PMID:22549784

  9. Serum amyloid A binds specific extracellular matrix glycoproteins and induces the adhesion of resting CD4+ T cells.

    PubMed

    Preciado-Patt, L; Hershkoviz, R; Fridkin, M; Lider, O

    1996-02-01

    Serum amyloid A (SAA), a prototypic acute phase protein reactant, exists naturally in the serum of healthy individuals. However, the levels of SAA in serum and its presence in sites of inflammation increase during certain chronic diseases associated with a local elevation of cytokine concentrations. Although the chemical structure of SAA is defined, its putative immunologic role(s) is still obscure. Nevertheless, it has been shown that 1) SAA acts as a chemoattractant and regulator of the migration of monocytes, polymorphonuclear cells, and T lymphocytes through endothelial cell monolayers; and 2) SAA and its proteolytically degraded N-terminal amyloid A fragment contain an extracellular matrix (ECM)-related cell adhesion epitopes. Herein, we examined whether SAA can associate with specific ECM moieties, and whether immobilized SAA-ECM complexes affect T lymphocyte adhesion. Radiolabeled human rSAA ([125I]rSAA) interacted avidly (Kd = 10(-9) M) and transiently with intact ECM, laminin, and vitronectin, but not with fibronectin or collagen type II. The binding of [125I]rSAA to ECM and laminin was inhibited by unlabeled rSAA and by the AA fragment, but not by the C-terminal portion of SAA (amino acid residues 2-82 and 77-104, respectively). Upon interactions with SAA or amyloid A, immobilized ECM, laminin, and vitronectin induced the adhesion of resting human CD4+ T cells in an apparently beta 1-integrin-mediated manner. Thus, the ECM appears to serve as a temporary anchorage site for SAA and amyloid A, and these ECM-complexed molecules seem to be involved in regulating the recruitment and accumulation of immunocytes in extravascular inflammatory compartments. PMID:8557997

  10. Docking of the Periplasmic FecB Binding Protein to the FecCD Transmembrane Proteins in the Ferric Citrate Transport System of Escherichia coli▿

    PubMed Central

    Braun, Volkmar; Herrmann, Christina

    2007-01-01

    Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter for vitamin B12, consisting of the BtuF binding protein and the BtuCD transmembrane proteins (E. L. Borths et al., Proc. Natl. Acad. Sci. USA 99:16642-16647, 2002). Here, we examined the role of the residues predicted to be involved in salt-bridge formation between FecB and FecCD by substituting these residues with alanine, cysteine, arginine, and glutamate and by analyzing the citrate-mediated iron transport of the mutants. Replacement of E93 in FecB with alanine [FecB(E93A)], cysteine, or arginine nearly abolished citrate-mediated iron transport. Mutation FecB(E222R) nearly eliminated transport, and FecB(E222A) and FecB(E222C) strongly reduced transport. FecD(R54C) and FecD(R51E) abolished transport, whereas other R-to-C mutations in putative interaction sites between FecCD and FecB substantially reduced transport. The introduced cysteine residues in FecB and FecCD also served to examine the formation of disulfide bridges in place of salt bridges between the binding protein and the transmembrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggest cross-linking of FecB(E93C) to FecD(R54C) and FecB(E222C) to FecC(R60C). The data are consistent with the proposal that FecB(E93) is contained in the region that binds to FecD and FecB(E222) in the region that binds to FecC. PMID:17660286

  11. CD4 binding to major histocompatibility complex class II antigens induces LFA-1-dependent and -independent homotypic adhesion of B lymphocytes.

    PubMed

    Kansas, G S; Cambier, J C; Tedder, T F

    1992-01-01

    T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag-presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4-gamma 3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4-gamma 3-induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre-B cells did not exhibit adhesion in response to these stimuli, suggesting that cross-linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II-induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA-D or HLA-DQ gene products induced adhesion. Anti-class II mAb and CD4-gamma 3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA-D antigens induced homotypic adhesion that was dependent on the activation of LFA-1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA-1-deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which

  12. CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells

    PubMed Central

    1996-01-01

    The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins. PMID:8551221

  13. A Stretch of Polybasic Residues Mediates Cdc42 GTPase-activating Protein (CdGAP) Binding to Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Its GAP Activity*

    PubMed Central

    Karimzadeh, Fereshteh; Primeau, Martin; Mountassif, Driss; Rouiller, Isabelle; Lamarche-Vane, Nathalie

    2012-01-01

    The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions. PMID:22518840

  14. The cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine inhibits LPS signaling by competing with endotoxin for CD14 binding.

    PubMed

    Piazza, Matteo; Calabrese, Valentina; Baruffa, Chiara; Gioannini, Theresa; Weiss, Jerrold; Peri, Francesco

    2010-12-15

    The identification of the bacterial endotoxin receptors for innate immunity, most notably the Toll-like receptor 4 (TLR4), has sparked great interest in therapeutic manipulation of innate immune system. We have recently developed synthetic molecules that have been shown to inhibit TLR4 activation in vitro and in vivo. Here we present the synthesis and the biological characterization of a new molecule, the cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine, with a structure strictly related to the previously developed TLR4 modulators. This compound is able to inhibit in a dose-dependent manner the LPS-stimulated TLR4 activation in HEK cells. In order to characterize the mechanism of action of this compound, we investigated possible interactions with the extracellular components that bind and shuttle LPS to TLR4, namely LBP, CD14, and MD-2. This compound inhibited LBP/CD14-dependent LPS transfer to MD-2.TLR4, resulting in reduced formation of a (LPS-MD-2-TLR4)(2) complex. This effect was due to inhibition of the transfer of LPS from aggregates in solution to sCD14 with little or no effect on LPS shuttling from LPS/CD14 to MD-2. This compound also inhibited transfer of LPS monomer from full-length CD14 to a truncated, polyhistidine tagged CD14. Taken together, our findings strongly suggest that this compound inhibits LPS-stimulated TLR4 activation by competitively occupying CD14 and thereby reducing the delivery of activating endotoxin to MD-2.TLR4.

  15. 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)

  16. 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.

  17. 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. PMID:17925391

  18. The Simpson-Golabi-Behmel syndrome causative glypican-3, binds to and inhibits the dipeptidyl peptidase activity of CD26.

    PubMed

    Davoodi, Jamshid; Kelly, John; Gendron, Nathalie H; MacKenzie, Alex E

    2007-06-01

    Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition shown to be the result of deletions of the glypican-3 (GPC3) gene. GPC3 is a proteoglycan localized to the cell membrane via a glycosylphosphatidyl-inositol (GPI) anchor. To further elucidate the GPC3 function(s), we have screened various cell lines for proteins that interact with GPC3, resulting in the isolation of a 115 kDa protein, identified as CD26. The interaction occurred with both the glycosylated and unglycosylated forms of GPC3 and led to the inhibition of CD26 peptidase activity. Moreover, introduction of CD26 into Cos-1 cells was accompanied by the up-regulation of cell growth, while inclusion of recombinant GPC3 in the media reduced the growth of CD26 transfected Cos-1 cells, drastically. Furthermore, HepG2 C3A cells containing CD26 underwent apoptosis in the presence of recombinant GPC3 in both concentration and time-dependant manner. In light of the fact that inhibition of CD26 reduces the rate of cell proliferation, we propose that a number of physical findings observed in SGBS patients may be a consequence of a direct interaction of GPC3 with CD26. Furthermore, GPC3 without the GPI anchor is capable of inducing apoptosis indicating that neither the GPI anchor nor the membrane attachment is required for apoptosis induction.

  19. 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

  20. 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.

  1. Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

    PubMed

    Caldas, Cristina; Coelho, Verônica; Kalil, Jorge; Moro, Ana Maria; Maranhão, Andrea Q; Brígido, Marcelo M

    2003-05-01

    Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

  2. Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

    PubMed Central

    Suzuki, Takashi; Suzuki, Miho; Ogino, Shinji; Umemoto, Ryo; Nishida, Noritaka; Shimada, Ichio

    2015-01-01

    CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions. PMID:26038553

  3. 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.

  4. Quantitative analysis of C4Ab and C4Bb binding to the C3b/C4b receptor (CR1, CD35)

    PubMed Central

    REILLY, B D; MOLD, C

    1997-01-01

    Complement-dependent clearance of immune complexes in humans is dependent on the activation and binding of the early components of the classical complement cascade. This prevents immune complex precipitation and promotes binding of the complexes by the C4b/C3b complement receptor CR1 (CD35) found on erythrocytes. The fourth component of human complement is encoded by two closely linked genes within the MHC. These genes give rise to the isotypic forms C4A and C4B, and recent studies suggest that CR1 binds activated C4A (C4Ab) to a greater extent than activated C4B (C4Bb). To study this difference in a more quantitative way the binding reactions between CR1 and C4Ab- and C4Bb-coated immune complexes and between CR1 and soluble dimers of C4Ab (C4Ab2) and C4Bb (C4Bb2) were analysed using the native receptor on human erythrocytes. The binding reaction between immune complexes with equivalent amounts of covalently bound C4Ab or C4Bb and erythrocyte CR1 showed a two-fold higher binding of complexes coated with C4A. Furthermore, erythrocyte CR1 bound C4Ab2 with an apparent four-fold higher affinity (Kd ≍ 1.4 10−7M) than C4Bb2 (Kd ≍ 4–8 10−7M), indicating a preferential binding of CR1 for C4A. PMID:9367418

  5. Colocalization of a CD1d-Binding Glycolipid with a Radiation-Attenuated Sporozoite Vaccine in Lymph Node-Resident Dendritic Cells for a Robust Adjuvant Effect.

    PubMed

    Li, Xiangming; Kawamura, Akira; Andrews, Chasity D; Miller, Jessica L; Wu, Douglass; Tsao, Tiffany; Zhang, Min; Oren, Deena; Padte, Neal N; Porcelli, Steven A; Wong, Chi-Huey; Kappe, Stefan H I; Ho, David D; Tsuji, Moriya

    2015-09-15

    A CD1d-binding glycolipid, α-Galactosylceramide (αGalCer), activates invariant NK T cells and acts as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying nearly 100-fold stronger CD1d binding affinity. In the current study, 7DW8-5 was found to exert a more potent adjuvant effect than αGalCer for a vaccine based on radiation-attenuated sporozoites of a rodent malaria parasite, Plasmodium yoelii, also referred to as irradiated P. yoelii sporozoites (IrPySpz). 7DW8-5 had a superb adjuvant effect only when the glycolipid and IrPySpz were conjointly administered i.m. Therefore, we evaluated the effect of distinctly different biodistribution patterns of αGalCer and 7DW8-5 on their respective adjuvant activities. Although both glycolipids induce a similar cytokine response in sera of mice injected i.v., after i.m. injection, αGalCer induces a systemic cytokine response, whereas 7DW8-5 is locally trapped by CD1d expressed by dendritic cells (DCs) in draining lymph nodes (dLNs). Moreover, the i.m. coadministration of 7DW8-5 with IrPySpz results in the recruitment of DCs to dLNs and the activation and maturation of DCs. These events cause the potent adjuvant effect of 7DW8-5, resulting in the enhancement of the CD8(+) T cell response induced by IrPySpz and, ultimately, improved protection against malaria. Our study is the first to show that the colocalization of a CD1d-binding invariant NK T cell-stimulatory glycolipid and a vaccine, like radiation-attenuated sporozoites, in dLN-resident DCs upon i.m. conjoint administration governs the potency of the adjuvant effect of the glycolipid. PMID:26254338

  6. Gill diseases of cultured salmonids in Ontario.

    PubMed Central

    Daoust, P Y; Ferguson, H W

    1983-01-01

    Between 1977 and 1981, the Fish Pathology Laboratory of the Ontario Veterinary College received 239 cases from trout farms of southern Ontario, 51 (21.3%) of which had diseased gills. Branchial lesions in 86.3% of these 51 cases were characterized by marked lamellar epithelial hyperplasia with epithelial hypertrophy and lamellar fusion. Filamentous bacteria were seen on the surface of the branchial filaments and lamellae in 68.6% of the cases. Our observations highlight the importance of gill diseases as a production problem of farmed salmonids in southern Ontario. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:6416657

  7. Control of the binding energy by tuning the single dopant position, magnetic field strength and shell thickness in ZnS/CdSe core/shell quantum dot

    NASA Astrophysics Data System (ADS)

    Talbi, A.; Feddi, E.; Zouitine, A.; Haouari, M. El; Zazoui, M.; Oukerroum, A.; Dujardin, F.; Assaid, E.; Addou, M.

    2016-10-01

    Recently, the new tunable optoelectronic devices associated to the inclusion of the single dopant are in continuous emergence. Combined to other effects such as magnetic field, geometrical confinement and dielectric discontinuity, it can constitute an approach to adjusting new transitions. In this paper, we present a theoretical investigation of magnetic field, donor position and quantum confinement effects on the ground state binding energy of single dopant confined in ZnS/CdSe core/shell quantum dot. Within the framework of the effective mass approximation, the Schrödinger equation was numerically been solved by using the Ritz variational method under the finite potential barrier. The results show that the binding energy is very affected by the core/shell sizes and by the external magnetic field. It has been shown that the single dopant energy transitions can be controlled by tuning the dopant position and/or the field strength.

  8. Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

    SciTech Connect

    Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

    1988-09-01

    Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing /sup 125/I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.

  9. The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

    PubMed

    Panni, Simona; Salvioli, Stefano; Santonico, Elena; Langone, Francesca; Storino, Francesca; Altilia, Serena; Franceschi, Claudio; Cesareni, Gianni; Castagnoli, Luisa

    2015-02-01

    Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.

  10. The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

    PubMed

    Panni, Simona; Salvioli, Stefano; Santonico, Elena; Langone, Francesca; Storino, Francesca; Altilia, Serena; Franceschi, Claudio; Cesareni, Gianni; Castagnoli, Luisa

    2015-02-01

    Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R. PMID:25261582

  11. Synthesis of 1-boraadamantaneamine derivatives with selective astrocyte vs C6 glioma antiproliferative activity. A novel class of anti-hepatitis C agents with potential to bind CD81.

    PubMed

    Wagner, Carl E; Mohler, Michael L; Kang, Gyong Suk; Miller, Duane D; Geisert, Eldon E; Chang, Yu-An; Fleischer, Everly B; Shea, Kenneth J

    2003-07-01

    A variety of amine complexes with 1-boraadamatane were synthesized and subsequently evaluated for an antiproliferative effect on CD81-enriched cell lines to provide evidence for binding and activation of CD81. CD81 is a member of the tetraspanin family of membrane proteins found in all cell lineages in the liver. CD81 signals for antiproliferation when bound by antibodies. It is known that the HCV-E2 envelope glycoprotein binds to the CD81 protein. While it is unclear whether virus entry into host cells is directly linked to virus attachment via CD81 for HCV, this step in the viral life cycle has recently proven to be an effective point of attack for other viruses including HIV and rhinoviruses. The aim of the current study concerns the synthesis of amantidine analogues by appending primary amines to 1-boraadamantane to evaluate such compounds for CD81-dependent antiproliferation of CD81-enriched cell lines (astrocyte) vs CD81-deficient cell lines (C6 glioma). If the antiproliferative effect of these amantidine analogues proves to be an effect of binding and activating CD81, then these compounds may have the potential to prevent or treat HCV infections. Each compound's potential for preventive and therapeutic activity stems from the compound's potential to block viral attachment, virus-cell fusion, or virus entry into host cells or to counter potential mechanisms of HCV immune evasion. Out of a library of over 500 compounds, including randomly selected small molecules and rationally designed small molecules, only the 1-boraadamantaneamine compounds and structurally similar analogues display a significant antiproliferative effect on the CD81-enriched astrocytes relative to the CD81-deficient cell lines. In fact, 1-boraadamantane.l-phenylalanine methyl ester complex (5), 1-boraadamantane.ethanolamine complex (8), and (S)-2-[(adamantane-1-carbonyl)amino]-3-phenylpropionic acid (15) show a dose-dependent, astrocyte-selective antiproliferative activity in the

  12. 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.

  13. Learning to Teach--Gill's Story

    ERIC Educational Resources Information Center

    Hatch, Gill; Rowland, Tim

    2006-01-01

    Gill Hatch was a very fine mathematician. Indeed, following her undergraduate studies in Cambridge in the late 1950s, she was one of the elite who went on to the notoriously difficult Part III of the Mathematical Tripos. In this article, the author describes the autobiographical accounts of Hatch during her teaching career in teacher education, as…

  14. 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.

  15. Characterization of the species-dependent ketoprofen/albumin binding modes by induced CD spectroscopy and TD-DFT calculations.

    PubMed

    Tedesco, Daniele; Pistolozzi, Marco; Zanasi, Riccardo; Bertucci, Carlo

    2015-08-10

    The stereospecificity of high-affinity biorecognition phenomena at the basis of the activity of drugs is an important topic of active research in medicinal chemistry. The binding of drugs to their targets or to carrier proteins may lead to the onset of an induced circular dichroism (ICD) signal, which can be detected experimentally. Quantum mechanical (QM) calculations based on density functional theory (DFT) and its time-dependent formulation (TD-DFT) can be used to determine the theoretical chiroptical response of all the possible conformations of drugs bound to their hosts; by comparison with the experimental ICD spectra of drug-host complexes, this approach can lead to the identification of possible binding modes in the absence of X-ray crystallography or NMR data. The present article reports the application of experimental electronic circular dichroism (ECD) spectroscopy, DFT conformational analysis and TD-DFT calculations to the investigation of the binding modes of (S)-ketoprofen to serum albumins. The peculiar species-dependent ICD spectra observed for the binding of (S)-ketoprofen to different serum albumins can be explained by the selection of different mutual arrangements of the phenyl moieties inside the binding pocket. Such structural elucidations contribute to a better understanding of the changes in the pharmacokinetic and pharmacodynamic profiles of drugs among different species.

  16. Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

    PubMed Central

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J.; Walker, Laura M.; Ramos, Alejandra; Diwanji, Devan C.; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S.; Stanfield, Robyn L.; McBride, Ryan; Marozsan, Andre J.; Paulson, James C.; Sanders, Rogier W.; Moore, John P.; Burton, Dennis R.; Poignard, Pascal; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  17. Plasmodium chabaudi-Infected Erythrocytes Adhere to CD36 and Bind to Microvascular Endothelial Cells in an Organ-Specific Way

    PubMed Central

    Mota, Maria M.; Jarra, William; Hirst, Elizabeth; Patnaik, Pradeep K.; Holder, Anthony A.

    2000-01-01

    Adherence of erythrocytes infected with Plasmodium falciparum to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. However, the real importance of sequestration for infection and disease has never been fully assessed. The absence of an appropriate in vivo model for sequestration has been a major barrier. We have examined the rodent malaria parasite Plasmodium chabaudi chabaudi AS in mice as a potential model. Erythrocytes infected with this parasite adhere in vitro to purified CD36, a critical endothelium receptor for binding P. falciparum-infected erythrocytes. P. c. chabaudi-infected erythrocytes adhere in vitro to endothelial cells in a gamma interferon-dependent manner, suggesting the involvement of additional adhesion molecules in the binding process, as is also the case with P. falciparum-infected cells. Furthermore, plasma or sera from infected and hyperimmune mice, respectively, have the ability to block binding of infected erythrocytes to endothelial cells. In vivo, erythrocytes containing mature P. c. chabaudi parasites are sequestered from the peripheral circulation. Sequestration is organ specific, occurring primarily in the liver, although intimate contact between infected erythrocytes and endothelial cells is also observed in the spleen and brain. The results are discussed in the context of the use of this model to study (i) the relationship between endothelial cell activation and the level of sequestration and (ii) the primary function of sequestration in malaria infection. PMID:10858230

  18. Joint effects of crude oil and heavy metals on the gill filament EROD activity of marbled rockfish Sebastiscus marmoratus.

    PubMed

    Zheng, Ronghui; Chen, Huanbin; Bo, Jun; Xie, Qing; Hong, Fukun; Zhang, Yusheng

    2016-10-01

    The aim of this study was to characterize dose- and time-dependent responses of gill 7-ethoxyresorufin O-deethylase (EROD) activity from Juvenile marbled rockfish (Sebastiscus marmoratus) exposed to the water-accommodated fraction (WAF) of crude oil and heavy metal Cd(Ⅱ) or Pb(Ⅱ) alone or in mixture. Compared to the control group, gill filament EROD activity in S. marmoratus was significantly induced after exposure to the WAF from 80 to 320μg/L for 5 days in dose response experiment and after exposure to 40μg/L WAF for 6-10 days in time course experiment, respectively. In the other hand, gill filament EROD activity were not significantly affected compared to the control group or related WAF groups no matter in the dose response experiment or in the time course experiment of Cd(Ⅱ), Pb(Ⅱ) or its mixture with WAF. The results suggest the use of gill filament EROD activity as a biomarker of exposure to waterborne AhR agonists in marine ecosystems while simultaneously being exposed to environmental concentrations of Cd(Ⅱ) or Pb(Ⅱ). PMID:27290642

  19. 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

  20. Optimal lamellar arrangement in fish gills

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

    We present the results of a combined theoretical and experimental investigation of the oxygen transport in fish gills. Efficient respiration is crucial to fish because of relatively low oxygen contents in water compared to that in air. Ordered structures of lamellae of fish gills offer extended surfaces for oxygen transport. While the more compact arrangement of the lamellae provides larger surface area for oxygen diffusion, it causes higher viscous resistance to water flow through the interlamellar space. This allows us to expect the optimal lamellar arrangement for maximizing the oxygen transport. By developing a dynamic model for oxygen transport in fish gills, we calculate optimal lamellar arrangement for maximizing oxygen transport. We demonstrate that the interlamellar distance of a broad range of fish species is consistent with the deduced optimal lamellar arrangement. Our results thus provide the first rationale for the relatively uniform interlamellar distance of many fish regardless of their size, appearance, and habitat. This work was supported by the Sogang Uinversity Research Grant of 2013 (201310009.01) and the National Research Foundation, Korea (2013034978).

  1. [Tics and Gilles de la Tourette syndrome].

    PubMed

    de Mattos, J P; de Rosso, A L

    1995-03-01

    The concept of tic was developed at the end of the XIX century, emerging from the "chaos of choreas". Tic is defined as involuntary contractions of agonist and antagonist muscles in one or more parts of the body. It can be suppressed by voluntary efforts for seconds or hours, followed by exacerbations. Gilles de la Tourette's original article was published in 1885, in which he described nine patients with tics, and vocalisations. The pathogenesis of Gilles de la Tourette syndrome remained obscure. However, three factors have been considered: the neurochemical factor, related to the increased dopaminergic activity at the basal ganglia; the genetic factor and the non-genetic factors, for which environment more than genetic factors are involved. Pathologic examinations failed to reveal structural lesions, but PET studies showed metabolic hypofunction on the frontal, cingulate and possibly insular cortex, and on the inferior corpus striatum. The motor tics as well as the vocal tics can be simple or complex and are present in all patients. Other signs can be added to the previous tics: sensory tics, echophilia, coprophilia, obsessions, compulsions and impulsions. Diagnostic criteria of Gilles de la Tourette syndrome are based on: age of onset; presence of motor and vocal tics; voluntary suppression of the movements; variation in number, type, location and severity of tics; duration of more than one year. Haloperidol is the drug of choice for the treatment of Tourette's syndrome.

  2. Histopathological changes in gill and liver of Capoeta capoeta living in the Karasu River, Erzurum.

    PubMed

    Dane, Hatice; Şişman, Turgay

    2015-07-01

    The contamination of surface waters by different pollutants is an important problem worldwide. In this study, the histopathological effects of water pollution were investigated on freshwater fish species Capoeta capoeta caught from the Karasu River. Fish were caught at three different sites in the Karasu River, namely, Aşkale, Dumlu, and Serçeme. The histological changes in gill and liver of fish were detected microscopically and evaluated with quantitative analyses. In addition, heavy metals have also been determined in surface water samples from these sites. Results showed that the Aşkale site was polluted by different kinds of heavy metals. In Aşkale site, some heavy metals such as Cd, Al, As, Pb, and Mn levels were mostly detected at concentrations above than the accepted values by the Turkish Standards Institute. The presence of gill and liver histological alterations were assessed by the degree of tissue change (DTC). In gill filaments, hypertrophy and hyperplasia of the gill epithelium, lamellar epithelial lifting, lamellae shortening, vasodilatation, lamellar disorganization, blood congestion, fusion, and aneurysm were observed. In the liver, the changes included an increase in the number and size of melanomacrophage aggregates, nonhomogenous parenchyma, proliferation of the hepatopancreas, sinusoidal dilatation, vacuolization, hypertrophy of the hepatocytes, congestion and degeneration of central vein, blood congestion, pyknotic nucleus, focal necrosis, and hepatic granuloma. The histological lesions were comparatively most severe in liver. The DTC means were varied from slight to moderate of gill and moderate to severe of liver tissue in the Aşkale site, thus the site is considered to be of low quality. Some pathological alterations were observed in the Serçeme site, although their distribution was lower than sites Dumlu and especially Aşkale. The least DTC means of the Serçeme site demonstrated their good environmental quality. The results

  3. 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.

  4. Assignment of the DPP4 gene encoding adenosine deaminase binding protein (CD26/dipeptidylpeptidase IV) to 2q23

    SciTech Connect

    Mathew, S.; Morrison, M.E.; Murty, V.V.V.S.

    1994-07-01

    FISH was performed on chromosome preparations obtained from phytohemagglutinin-stimulated human blood lymphocytes. cDNA encoding ADAbp was isolated from the SK-RC-28 human renal cell carcinomas cell line using PCR technique and was cloned in pSVK3 plasmid for use as a probe. The PCR primers were constructed from the known nucleotide sequence of CD26, and the cDNA product was extracted from nucleotides 1 to 2344. The vector containing the probe was labeled by nick-translation with biotin-11-dUTP. Hybridization to chromosome spreads, washings, detection with FITC-conjugated avidin, selection and photography of metaphases, analysis of signals, and banding were performed according to the described method.

  5. 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.

  6. 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 Central

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

    2013-01-01

    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 β-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) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly fold roll of the Hcn sub-domain. The PG4 moeity 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 molecule was observed bound to the equivalent residues suggesting that

  7. Simple and sensitive progesterone detection in human serum using a CdSe/ZnS quantum dot-based direct binding assay.

    PubMed

    Oh, Sung-Duk; Duong, Hong Dinh; Rhee, Jong Il

    2015-08-15

    In this study, we developed a CdSe/ZnS quantum dot (QD)-based immunoassay for use in determining the presence of progesterone (P4) in human serum. Hydrophilic QDs were conjugated to anti-progesterone antibody (P4Ab) via ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. After purification, the P4Ab-QD conjugates were immobilized onto the wells of a 96-well microtiter plate, and a direct-binding immunoassay based on the binding of P4 to immobilized P4Ab-QD conjugates had a detection limit of 0.21 ng/ml and a sensitivity of 1.37 ng/ml, with a linear range of 0.385 to 4.55 ng/ml. The proposed immunoassay was successfully used to determine the P4 concentration in real human serum, and the results showed a good correlation with the accredited radioimmunoassay (RIA). PMID:25963894

  8. 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.

  9. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  10. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  11. 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.

  12. 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. PMID:15713467

  13. Cloning of two members of the SIRP alpha family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells.

    PubMed

    Brooke, G P; Parsons, K R; Howard, C J

    1998-01-01

    Recent experimental studies have greatly clarified the function of cell surface molecules in the induction and modulation of T cell responses by antigen-presenting cells (APC). However, the differences in ability to stimulate T cells evident for different types and subpopulations of the same APC, such as dendritic cell subsets, is less well understood. This report details an investigation of an antigen expressed on monocytes that is also expressed on a subset of cattle afferent lymph veiled cells (ALVC). A cDNA library derived from cattle monocytes was screened with monoclonal antibodies (mAb) for expression in COS-7 cells. Using separate mAb for screening, two cDNA were cloned, the sequences of which showed a single long open reading frame encoding a predicted type I glycoprotein of 506 amino acids that contained three immunoglobulin superfamily domains and a long 112-amino acid cytoplasmic tail. We have termed this antigen MyD-1, reflecting its myeloid and dendritic cell distribution. Analysis of the EMBL database revealed that the molecule is a member of the recently described family of signal regulatory proteins (SIRP). The outeremost Ig domain was of the adhesion/receptor I-type, suggesting that MyD-1 might bind to a ligand on another cell. Evidence for this was subsequently obtained by demonstrating that COS-7 cells transfected with MyD-1 cDNA bound CD4 T cells and this binding was blocked by specific mAb. The potential importance of this interaction was supported by the finding that the proliferation of resting memory CD4 T cells to ovalbumin-pulsed monocytes was significantly reduced in the presence of mAb to MyD-1. A role for the molecule in the modulation of the monocyte/dendritic APC response is also predicted from the existence of multiple potential tyrosine phosphorylation sites in the cytoplasmic domain, including the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the observation that the SIRP alpha family members have been

  14. 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.

  15. 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.

  16. 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

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

    PubMed

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

    2015-12-01

    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.

  18. The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidized low-density lipoprotein.

    PubMed Central

    Malaud, Eric; Hourton, Delphine; Giroux, Louise Marie; Ninio, Ewa; Buckland, Robin; McGregor, John L

    2002-01-01

    CD36, a major adhesion molecule expressed by monocytes/macrophages, plays a key role in the binding and internalization of oxidized low-density lipoprotein (OxLDL). This adhesion molecule, a member of an important scavenger receptor family, contains a very short C-terminal cytoplasmic tail that is known to induce intracellular signalling events. However, the domains on the cytoplasmic tail involved in such signal transduction are unknown. In this study, we have investigated the functional components of the cytoplasmic tail by site-directed mutagenesis coupled with functional OxLDL and monoclonal antibody (mAb) binding studies. Seven truncated or punctual CD36 constructs, localized in the cytoplasmic tail, were produced by site-directed mutagenesis. Each construct was stably expressed in HEK293 cells. We used a quantitative and a qualitative method, labelling OxLDL with either iodine or rhodamine, to determine the functional importance of the cytoplasmic domains in OxLDL internalization. Results indicate that: (1) a deletion of the last amino-acid (construct K472STOP) significantly reduces, compared with wild-type, the binding, internalization and degradation of OxLDL; (2) truncation of the last six amino-acids (construct R467STOP) significantly reduces OxLDL binding; (3) the above two constructs (K472STOP and R467STOP) showed a reduced rate of OxLDL internalization compared with wild-type; (4) the binding and rate of internalization of an anti-CD36 monoclonal antibody (10/5) was not affected by the above mentioned mutants (K472STOP and R467STOP), compared with wild-type. This study shows, for the first time, a specific site on the CD36 cytoplasmic tail that is critical for the binding, endocytosis and targeting of OxLDL. PMID:12023894

  19. HbAHP-25, an In-Silico Designed Peptide, Inhibits HIV-1 Entry by Blocking gp120 Binding to CD4 Receptor

    PubMed Central

    Bashir, Tahir; Patgaonkar, Mandar; Kumar C, Selvaa; Pasi, Achhelal; Reddy, Kudumula Venkata Rami

    2015-01-01

    Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection. PMID:25915507

  20. HbAHP-25, an In-Silico Designed Peptide, Inhibits HIV-1 Entry by Blocking gp120 Binding to CD4 Receptor.

    PubMed

    Bashir, Tahir; Patgaonkar, Mandar; Kumar, Selvaa C; Pasi, Achhelal; Reddy, Kudumula Venkata Rami

    2015-01-01

    Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection. PMID:25915507

  1. Oculomotor control in Gilles de la Tourette syndrome.

    PubMed Central

    Bollen, E L; Roos, R A; Cohen, A P; Minderaa, R B; Reulen, J P; Van de Wetering, B J; Van Woerkom, T C; Buruma, O J

    1988-01-01

    Saccadic eye movements, fixation and smooth pursuit were studied in 28 children with Gilles de la Tourette syndrome and found to be normal. A link has been postulated between Gilles de la Tourette syndrome and other movement disorders. The results obtained in the present series do not support this hypothesis. PMID:3216209

  2. 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

  3. Iris Pigment Epithelium Expressing CD86 (B7-2) Directly Suppresses T Cell Activation In Vitro via Binding to Cytotoxic T Lymphocyte–associated Antigen 4

    PubMed Central

    Sugita, Sunao; Streilein, J. Wayne

    2003-01-01

    A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation. PMID:12835481

  4. 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.

  5. 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. PMID:25403762

  6. 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.

  7. Transcriptome profiling analysis of rare minnow (Gobiocypris rarus) gills after waterborne cadmium exposure.

    PubMed

    Wang, Zhi-Jian; Liu, Xiao-Hong; Jin, Li; Pu, De-Yong; Huang, Jing; Zhang, Yao-Guang

    2016-09-01

    Rare minnow (Gobiocypris rarus) is a widely used experimental fish in risk assessments of aquatic pollutants in China. Cadmium (Cd) is one of the most toxic heavy metals in the world; however, few studies have used fish gills, a multi-functional organ. In this study, we characterized the differential expression of adult female rare minnow gills after sub-chronic waterborne Cd (75μg/L CdCl2) exposure for 35d. A total of 452 genes (209 up-regulated and 243 down-regulated) were identified by gene expression profiling using RNA-Seq before and after treatment. Of these differentially expressed genes, 75, 21, and 54 differentially expressed genes are related to ion transport, oxidation-reduction processes, and the immune response, respectively. The results of GO and KEGG enrichment analyses, together with the altered transcript levels of major histocompatibility complex (MHC) class I and class II molecules and the significant increases in the levels of serum tumor necrosis factor α (TNF-α), interleukin 1β (IL1β) and nuclear factor-κB (NF-κB), indicated a disruption of the immune system, particularly the induction of inflammation and autoimmunity. The significant down-regulation of coagulation factor XIII A1 polypeptide (F13A1), tripartite motif-containing protein 21 (TRIM21), and Golgi-associated plant pathogenesis-related protein (GAPr) during both acute (≤96h) and sub-chronic (35d) waterborne Cd exposure, as well as their dosage dependence, suggested that these three genes could be used as sensitive biomarkers for aquatic Cd risk assessment. PMID:27292131

  8. [Gilles-de-la-Tourette syndrome].

    PubMed

    Schauenburg, H; Dressler, D

    1992-08-01

    Gilles de la Tourette's syndrome, a combination of multiple chronic tics and vocalizations, usually first occurring during childhood, is described in its history, symptomatology, genetics, etiology and therapy. Traditionally TS has been viewed either as an organic or as a psychogenic disorder. We propose an integrative concept combining both aspects. During a vulnerable phase in childhood a hypersensitivity of dopamine 2-receptors, induced by gene defects or perinatal trauma, leads to a lack of suppression of subcortical programs which discharge as tics. Tics are modified by multiple psychological contents (aggressive or sexual impulses, imitation of others) which tend to become independent of their origin. Severity of tics in the course of the illness is often dependent on the emotional status of the patient. Recent research focuses on the search for a major gene locus and the relationship between dopamine-receptor hypersensibility and the disturbances of other neurotransmitter systems (norepinephrine, serotonin, endorphin). PMID:1522932

  9. [Gilles-de-la-Tourette syndrome].

    PubMed

    Schauenburg, H; Dressler, D

    1992-08-01

    Gilles de la Tourette's syndrome, a combination of multiple chronic tics and vocalizations, usually first occurring during childhood, is described in its history, symptomatology, genetics, etiology and therapy. Traditionally TS has been viewed either as an organic or as a psychogenic disorder. We propose an integrative concept combining both aspects. During a vulnerable phase in childhood a hypersensitivity of dopamine 2-receptors, induced by gene defects or perinatal trauma, leads to a lack of suppression of subcortical programs which discharge as tics. Tics are modified by multiple psychological contents (aggressive or sexual impulses, imitation of others) which tend to become independent of their origin. Severity of tics in the course of the illness is often dependent on the emotional status of the patient. Recent research focuses on the search for a major gene locus and the relationship between dopamine-receptor hypersensibility and the disturbances of other neurotransmitter systems (norepinephrine, serotonin, endorphin).

  10. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

    PubMed

    Spruyt, L L; Glennie, M J; Beyers, A D; Williams, A F

    1991-12-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

  11. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16)

    PubMed Central

    1991-01-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i. PMID:1683892

  12. 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

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

    PubMed Central

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

    1986-01-01

    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) Images FIGURE 7. PMID:3709468

  14. 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

  15. The role of charge and multiple faces of the CD8 alpha/alpha homodimer in binding to major histocompatibility complex class I molecules: support for a bivalent model.

    PubMed Central

    Giblin, P A; Leahy, D J; Mennone, J; Kavathas, P B

    1994-01-01

    The CD8 dimer interacts with the alpha 3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8 alpha/alpha homodimer that are likely to be involved in binding to class I. Several key residues are situated on the top face of the dimer within loops analogous to the complementarity-determining regions (CDRs) of immunoglobulin. In addition, other important amino acids are located in the A and B beta-strands on the sides of the dimer. The potential involvement of amino acids on both the top and the side faces of the molecule is consistent with a bivalent model for the interaction between a single CD8 alpha/alpha homodimer and two class I molecules and may have important implications for signal transduction in class I-expressing cells. This study also demonstrates a role for the positive surface potential of CD8 in class I binding and complements previous work demonstrating the importance of a negatively charged loop on the alpha 3 domain of class I for CD8 alpha/alpha-class I interaction. We propose a model whereby residues located on the CDR-like loops of the CD8 homodimer interact with the alpha 3 domain of MHC class I while amino acids on the side of the molecule containing the A and B beta-strands contact the alpha 2 domain of class I. Images PMID:8127870

  16. Full characterization of the Cu-, Zn-, and Cd-binding properties of CnMT1 and CnMT2, two metallothioneins of the pathogenic fungus Cryptococcus neoformans acting as virulence factors

    PubMed Central

    Palacios, Òscar; Espart, Anna; Espín, Jordi; Ding, Chen; Thiele, Dennis J.; Atrian, Sílvia; Capdevila, Mercè

    2014-01-01

    We report here the full characterization of the metal binding abilities of CnMT1 and CnMT2, two Cryptococcus neoformans proteins recently identified as metallothioneins (MTs), which have been shown to perform a crucial role for the virulence and pathogenicity of this human-infecting fungus. In this work, we first performed a thorough in silico study of the CnMT1 and CnMT2 genes, cDNAs and corresponding encoded products. Subsequently, the Zn(II)-, Cd(II)- and Cu(I) binding abilities of both proteins were fully determined through the analysis of the metal-to-protein stoichiometries and the structural features (determined by ESI-MS, CD, ICP-AES and UV-vis spectroscopies) of the corresponding recombinant Zn-, Cd- and Cu-MT preparations synthesized in metal-enriched media. Finally, the analysis of the Zn/Cd and Zn/Cu replacement processes undertaken by the respective Zn-MT complexes when allowed to react with Cd(II) or Cu(I) aqueous solutions completed the analysis. Comprehensive consideration of all gathered results allow us to consider both isoforms as genuine copper-thioneins, and led to the identification of unprecedented Cu5-core clusters in MTs. CnMT1 and CnMT2 polypeptides appear as evolutionary related to the small fungal MTs, probably by ancient tandem-duplication events responding to a high selective pressure to chelate copper, and far from the properties of Zn- and Cd-thioneins. Finally, we propose a modular structure of the Cu-CnMT1 and Cu-CnMT2 complexes basically built on the three and five-fold presence of 7-Cys units, each one yielding a Cu5-core cluster. PMID:24317230

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

    PubMed Central

    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. PMID:25970007

  18. A mutation in the alpha 3 domain of Db that abrogates CD8 binding does not affect presentation of an immunodominant H-Y peptide.

    PubMed Central

    Dutz, J P; Teh, S J; Killeen, N; Teh, H S

    1995-01-01

    The peptidic nature of the male (H-Y) antigen, a model minor histocompatibility antigen in H-2b mice, has recently been demonstrated. In this study we show that the H-Y peptide, which is recognized by PM-1, a Db-restricted cytotoxic T-lymphocyte (CTL) clone, is absent in male H-2d spleen cells but present in male H-2d spleen cells that also express a transgenic Db molecule under its endogenous promoter. This result indicates that both the H-Y and the Db gene products are essential and sufficient for production of the Db-restricted H-Y peptide. By comparing the ability of the PM-1 clone and bulk CTL generated in a secondary mixed lymphocyte culture to recognize H-Y peptidic material eluted from affinity-purified Db molecules and separated by reversed-phase high-performance liquid chromatography (HPLC), we provide evidence that there is an immunodominant H-Y epitope that is presented by the Db molecule. Furthermore, the presentation of this epitope is not affected by a mutation in the alpha 3 domain of Db (asp227 to lys227), which abrogates CD8 binding, since similar amounts of H-Y peptide were eluted from affinity-purified wild-type or mutant Db molecules. However, the generation of the H-Y epitope is dependent on the presence of beta 2-microglobulin, since it is absent in male H-2b mice that lack a functional beta 2-microglobulin gene. The implications of these findings on T-cell development are discussed. PMID:7543449

  19. 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.

  20. 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.

  1. Effects of cadmium exposure on the gill proteome of Cottus gobio: modulatory effects of prior thermal acclimation.

    PubMed

    Dorts, Jennifer; Kestemont, Patrick; Thézenas, Marie-Laetitia; Raes, Martine; Silvestre, Frédéric

    2014-09-01

    Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. The aim of the present study was to investigate whether acclimation to elevated temperature affects the response of the European bullhead (Cottus gobio) to subsequent cadmium (Cd) exposure by using enzymatic and proteomic approaches. Fish acclimated to 15 (standard temperature), 18 or 21 °C for 28 days were exposed to 1mg Cd/L for 4 days at the respective acclimation temperature. First, exposure to Cd significantly decreased the activity of the lactate dehydrogenase (LDH) in gills of fish acclimated to 15 or 18 °C. However, an acclimation to 21 °C suppressed the inhibitory effect of Cd. Second, using a proteomic analysis by 2D-DIGE, we observed that thermal acclimation was the first parameter affecting the protein expression profile in gills of C. gobio, while subsequent Cd exposure seemed to attenuate this temperature effect. Moreover, our results showed opposite effects of these two environmental stressors at protein expression level. From the 52 protein spots displaying significant interaction effects of temperature and Cd exposure, a total of 28 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet algorithms of Scaffold software. The identified differentially expressed proteins can be categorized into diverse functional classes, related to protein turnover, folding and chaperoning, metabolic process, ion transport, cell signaling and cytoskeleton. Within a same functional class, we further reported that several proteins displayed reverse responses following sequential exposure to heat and Cd. This work provides insights into the molecular pathways potentially involved in heat acclimation process and the interactive effects of temperature and Cd stress in ectothermic vertebrates.

  2. 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)

  3. 18. Historic American Buildings Survey Gill and Mead, Architects 1907 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. Historic American Buildings Survey Gill and Mead, Architects 1907 ORIGINAL DRAWING OF SECOND FLOOR PLAN From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  4. 13. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects May 8, 1907 ORIGINAL DRAWING OF EAST ELEVATION From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  5. 20. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects September 20, 1907 ORIGINAL DRAWING OF ROOF DETAILS From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  6. [Accessory symptomatology and therapy of Gilles de la Tourette's disease].

    PubMed

    Achkova, M; Terziev, D

    1987-01-01

    Eight patients with Gilles de la Tourette's syndrome were examined. They had typical multiple tics and accessory disturbances--impulsive and reactive symptoms. The authors described the classification of accessory symptoms and the therapeutic approaches.

  7. 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. PMID:24979223

  8. 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.

  9. Local connected fractal dimension analysis in gill of fish experimentally exposed to toxicants.

    PubMed

    Manera, Maurizio; Giari, Luisa; De Pasquale, Joseph A; Sayyaf Dezfuli, Bahram

    2016-06-01

    An operator-neutral method was implemented to objectively assess European seabass, Dicentrarchus labrax (Linnaeus, 1758) gill pathology after experimental exposure to cadmium (Cd) and terbuthylazine (TBA) for 24 and 48h. An algorithm-derived local connected fractal dimension (LCFD) frequency measure was used in this comparative analysis. Canonical variates (CVA) and linear discriminant analysis (LDA) were used to evaluate the discrimination power of the method among exposure classes (unexposed, Cd exposed, TBA exposed). Misclassification, sensitivity and specificity, both with original and cross-validated cases, were determined. LCFDs frequencies enhanced the differences among classes which were visually selected after their means, respective variances and the differences between Cd and TBA exposed means, with respect to unexposed mean, were analyzed by scatter plots. Selected frequencies were then scanned by means of LDA, stepwise analysis, and Mahalanobis distance to detect the most discriminative frequencies out of ten originally selected. Discrimination resulted in 91.7% of cross-validated cases correctly classified (22 out of 24 total cases), with sensitivity and specificity, respectively, of 95.5% (1 false negative with respect to 21 really positive cases) and 75% (1 false positive with respect to 3 really negative cases). CVA with convex hull polygons ensured prompt, visually intuitive discrimination among exposure classes and graphically supported the false positive case. The combined use of semithin sections, which enhanced the visual evaluation of the overall lamellar structure; of LCFD analysis, which objectively detected local variation in complexity, without the possible bias connected to human personnel; and of CVA/LDA, could be an objective, sensitive and specific approach to study fish gill lamellar pathology. Furthermore this approach enabled discrimination with sufficient confidence between exposure classes or pathological states and avoided

  10. 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.

  11. 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. PMID:26418972

  12. 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

  13. 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.

  14. 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

  15. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay*

    PubMed Central

    AbuSamra, Dina B.; Al-Kilani, Alia; Hamdan, Samir M.; Sakashita, Kosuke; Gadhoum, Samah Z.; Merzaban, Jasmeen S.

    2015-01-01

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. PMID:26124272

  16. The interbranchial lymphoid tissue of Atlantic Salmon (Salmo salar L) extends as a diffuse mucosal lymphoid tissue throughout the trailing edge of the gill filament.

    PubMed

    Dalum, Alf S; Austbø, Lars; Bjørgen, Håvard; Skjødt, Karsten; Hordvik, Ivar; Hansen, Tom; Fjelldal, Per G; Press, Charles McL; Griffiths, David J; Koppang, Erling O

    2015-09-01

    The teleost gill forms an extensive, semipermeable barrier that must tolerate intimate contact with the surrounding environment and be able to protect the body from external pathogens. The recent discovery of the interbranchial lymphoid tissue (ILT) has initiated an anatomical and functional investigation of the lymphoid tissue of the salmonid gill. In this article, sectioning of gill arches in all three primary planes revealed an elongation of the ILT outward along the trailing edge of the primary filament to the very distal end, a finding not previously described. This newly found lymphoid tissue was investigated using a range of morphological and transcriptional tools. Avoiding potential salinity-related effects, the study focused on two fresh-water life stages-smoltifying juveniles and mature adults. Aggregates of T-cells continuous with the ILT were found within the thick epithelial lining of the trailing edge of the filament in considerably larger numbers than seen in the epithelium of the leading edge and of the interlamellar area. Only a few of these cells were identified as CD8α(+) -cells, and there was a significantly (P < 0.05) higher relative expression of CD4- than of CD8- related genes in all gill segments investigated. Numerous major histocompatibility complex class II(+) -cells were distributed uniformly throughout the filament epithelial tissue. Few Ig(+) -cells were detected. Overall, the morphological features and comparable immune gene expression of the previously described ILT and the filament trailing edge lymphoid tissue suggest a close functional and anatomical relationship. We propose that the anatomical definition of the ILT must be broadened to include both the previously described ILT (to be renamed proximal ILT) and the trailing edge lymphoid tissue (to be named distal ILT). This extended anatomical localisation identifies the ILT as a widely distributed mucosal lymphoid tissue in the gill of Atlantic salmon.

  17. 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

  18. 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.

  19. 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. PMID:26227807

  20. Extraction and metabolism of circulating catecholamines by the trout gill

    SciTech Connect

    Nekvasil, N.P.; Olson, K.R.

    1986-03-01

    Extraction and metabolism of (3H)-norepinephrine (NE) and (3H)epinephrine (E) by the respiratory (efferent branchial) and filamental (venous) vasculature of the trout gill were examined using an isolated perfused arch technique in which outflow from the two circulations was separated. Deaminated and O-methylated metabolites in the effluent were identified by ion-exchange chromatography. Metabolism by tissue homogenates was also measured. Gill homogenates deaminate catecholamines (CAs) faster than homogenates of liver, kidney, and skeletal muscle; branchial O-methylation was comparable to that of liver and kidney. The perfused gill extracted 60% of a NE pulse and 47% of an E pulse. During continuous CA perfusion the gill removed greater than 30% of the NE from the circulation through extraction and metabolism; only 7% of the E was removed. The gill venous system extracts and metabolizes more CAs than the efferent. NE is the preferred substrate for extraction and metabolism. A mechanism is proposed whereby high circulating CA levels, common during stress, are maintained through CA-induced reduction in venous blood flow. After the stress is alleviated, CA levels begin to fall, flow to the venous pathway increases, and the rate of inactivation of circulating CAs increases.

  1. Non-respiratory blood vessels in Latimeria gill filaments

    PubMed Central

    Vogel, W. O. P.

    1998-01-01

    A study of the blood pathways within the gills of Latimeria has been carried out using light and transmission electron microscopy. Clear evidence has been found for the presence of a secondary non-respiratory circulation in addition to the well-established respiratory pathway through the gill lamellae. All essential components of this system have been observed and have the same relationships and basic structure as comparable secondary systems in actinopterygian and elasmobranch fishes. These include a central venous sinus (CVS), arterio-venous anastomoses (AVAs) and central filament arteries (CFAs). AVAs connect both arterial vessels of the primary circulation and CFAs of the secondary circulation to the CVS. The latter contained many red blood cells. The presence of this secondary circulation in Latimeria gills contrasts with the situation in the gills of the three living genera of lungfishes where a system possessing the essential features of the tetrapod lymphatic vessel system has been recognized. No suggestions of a true lymphatic vessel system were observed in Latimeria. Other features of gill and vascular anatomy in Latimeria show its closer relationship to dipnoans than other groups of living fishes but evidence derived from this study of the secondary circulation clearly supports the view that the Dipnoi rather than Latimeria represent the living fishes most closely related to the tetrapods.

  2. 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.

  3. Biomolecular interaction study of hydralazine with bovine serum albumin and effect of β-cyclodextrin on binding by fluorescence, 3D, synchronous, CD, and Raman spectroscopic methods.

    PubMed

    Bolattin, Mallavva B; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

    2016-07-01

    Spectrofluoremetric technique was employed to study the binding behavior of hydralazine with bovine serum albumin (BSA) at different temperatures. Binding study of bovine serum albumin with hydralazine has been studied by ultraviolet-visible spectroscopy, fluorescence spectroscopy and confirmed by three-dimensional, synchronous, circular dichroism, and Raman spectroscopic methods. Effect of β-cyclodextrin on binding was studied. The experimental results showed a static quenching mechanism in the interaction of hydralazine with bovine serum albumin. The binding constant and the number of binding sites are calculated according to Stern-Volmer equation. The thermodynamic parameters ∆H(o) , ∆G(o) , ∆S(o) at different temperatures were calculated. These indicated that the hydrogen bonding and weak van der Waals forces played an important role in the interaction. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the donor (BSA) and acceptor (hydralazine) was evaluated and found to be 3.95 nm. Spectral results showed that the binding of hydralazine to BSA induced conformational changes in BSA. The effect of common ions on the binding of hydralazine to BSA was also examined. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Effects of environmental and physiological variables on the uptake of hydrophobic contaminants by the gills of rainbow trout, Salmo gairdneri (Richardson)

    SciTech Connect

    Black, M.C.

    1989-01-01

    This study investigated the effects of changes in contaminant uptake due to sorption to humic acid and changes in fish respiration, induced by altering respiratory demand via an acute change in temperature and by altering the diffusional capacity of the gill membrane by exposure to chlorine. In each experimental design the uptake of selected polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyl congeners (PCBs) by the gills of rainbow trout were measured simultaneously with measurements of fish respiratory functions using a fish metabolic chamber. Binding to humic acid (HA) reduced the bioavailability of benzo(a)pyrene (BaP) and 2,2{prime},5,5{prime}-tetrachlorobiphenyl (TCB). A 1:1 correlation was found between reductions in the percentage of BaP or TCB that was freely dissolved and reductions in uptake of the contaminant by trout gills. Eight to 24-h exposure to chlorine resulted in damage to gill lamellae. Reductions in the uptake efficiencies of oxygen and three PCB congeners in chlorine-induced damage to the gill membrane. However, increases in ventilatory functions compensated for the lowered uptake efficiencies and resulted in no net change in either oxygen or PCB uptake.

  5. Proteomic basis of stress responses in the gills of the pacific oyster Crassostrea gigas.

    PubMed

    Zhang, Yang; Sun, Jin; Mu, Huawei; Li, Jun; Zhang, Yuehuan; Xu, Fengjiao; Xiang, Zhiming; Qian, Pei-Yuan; Qiu, Jian-Wen; Yu, Ziniu

    2015-01-01

    The Pacific oyster Crassostrea gigas is one of the dominant sessile inhabitants of the estuarine intertidal zone, which is a physically harsh environment due to the presence of a number of stressors. Oysters have adapted to highly dynamic and stressful environments, but the molecular mechanisms underlying such stress adaptation are largely unknown. In the present study, we examined the proteomic responses in the gills of C. gigas exposed to three stressors (high temperature, low salinity, and aerial exposure) they often encounter in the field. We quantitatively compared the gill proteome profiles using iTRAQ-coupled 2-D LC-MS/MS. There were 3165 identified proteins among which 2379 proteins could be quantified. Heat shock, hyposalinity, and aerial exposure resulted in 50, 15, and 33 differentially expressed gill proteins, respectively. Venn diagram analysis revealed substantial different responses to the three stressors. Only xanthine dehydrogenase/oxidase showed a similar expression pattern across the three stress treatments, suggesting that reduction of ROS accumulation may be a conserved response to these stressors. Heat shock caused significant overexpression of molecular chaperones and production of S-adenosyl-l-methionine, indicating their crucial protective roles against protein denature. In addition, heat shock also activated immune responses, Ca(2+) binding protein expression. By contrast, hyposalinity and aerial exposure resulted in the up-regulation of 3-demethylubiquinone-9 3-methyltransferase, indicating that increase in ubiquinone synthesis may contribute to withstanding both the osmotic and desiccation stress. Strikingly, the majority of desiccation-responsive proteins, including those involved in metabolism, ion transportation, immune responses, DNA duplication, and protein synthesis, were down-regulated, indicating conservation of energy as an important strategy to cope with desiccation stress. There was a high consistency between the expression

  6. 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

  7. Applications of circular dichroism (CD) spectroscopy as educational tool for undergraduate students and monitoring protein conformational changes induced by ligand binding

    NASA Astrophysics Data System (ADS)

    Van Der Kar, Catherine Ann

    The work reported here provides an overview on the applications of circular dichroism (CD) spectroscopy for the study of protein secondary structure. This project has two specific goals. First, to develop and validate an undergraduate experiment for CHEM 3175 (biophysical chemistry laboratory) to demonstrate the use of CD spectroscopy for the determination of protein secondary structure. In an extension of this work, the same principles of analysis were applied to the study and characterization of the protein-ligand interaction of MiaE (a non-heme iron hydroxylase) and its substrate, tRNA.

  8. 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

  9. 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

  10. Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

    PubMed Central

    Pohlers, Dirk; Schmidt-Weber, Carsten B; Franch, Angels; Kuhlmann, Jürgen; Bräuer, Rolf; Emmrich, Frank; Kinne, Raimund W

    2002-01-01

    The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies. PMID:12010568

  11. A Membrane-bound Hemoglobin from Gills of the Green Shore Crab Carcinus maenas*

    PubMed Central

    Ertas, Beyhan; Kiger, Laurent; Blank, Miriam; Marden, Michael C.; Burmester, Thorsten

    2011-01-01

    Most hemoglobins serve for the transport or storage of O2. Although hemoglobins are widespread in “entomostracan” Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe2+ and an oxygen affinity of P50 = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O2 supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans. PMID:21118803

  12. Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

    PubMed

    Chalbi, Myriam; Barraud-Lange, Virginie; Ravaux, Benjamin; Howan, Kevin; Rodriguez, Nicolas; Soule, Pierre; Ndzoudi, Arnaud; Boucheix, Claude; Rubinstein, Eric; Wolf, Jean Philippe; Ziyyat, Ahmed; Perez, Eric; Pincet, Frédéric; Gourier, Christine

    2014-10-01

    Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

  13. The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits

    PubMed Central

    Hu, John C.; Greene, Christopher J.; King-Lyons, Natalie D.; Connell, Terry D.

    2015-01-01

    Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8+ T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8+ T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8+ T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants. PMID:26565800

  14. 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)

  15. A case of Gilles de la Tourette's syndrome

    PubMed Central

    Prakash, Jyoti; Singh, Pragnya; Bhat, P. S.; Srivastava, K.; Gupta, Vikash

    2015-01-01

    Gilles de la Tourette's syndrome is an uncommon illness associated with repetitive un-voluntary abnormal movements and utterance. It is often associated with other psychiatric morbidities. Management requires awareness of this uncommon illness, keen observation, relevant evaluation, and combination of pharmacology and psychotherapy for an optimal outcome. This case is brought out here for florid presentation and nuances of management. PMID:27212827

  16. Gilles de la Tourette syndrome in mental handicap.

    PubMed

    Reid, A H

    1984-03-01

    A case of Gilles de la Tourette syndrome in a mildly mentally retarded adult female is described. The clinical features, natural history and response to treatment were typical of the condition but the association with mental retardation, epilepsy and psychotic phenomena were unusual.

  17. 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)…

  18. Psychological Aspects of Gilles De La Tourette Syndrome.

    ERIC Educational Resources Information Center

    Grossman, Hildreth Youkilis; And Others

    1986-01-01

    Evaluated the psychopathological features that may underlie or accompany Gilles de la Tourette Syndrome. Univariate analyses indicated that Tourette subjects scored higher on the following scales of the Minnesota Multiphasic Personality Inventory: Schizophrenia, Depression, Psychopathic Deviate, Psychasthenia and Hypochondriasis. The results…

  19. A case of Gilles de la Tourette's syndrome.

    PubMed

    Prakash, Jyoti; Singh, Pragnya; Bhat, P S; Srivastava, K; Gupta, Vikash

    2015-01-01

    Gilles de la Tourette's syndrome is an uncommon illness associated with repetitive un-voluntary abnormal movements and utterance. It is often associated with other psychiatric morbidities. Management requires awareness of this uncommon illness, keen observation, relevant evaluation, and combination of pharmacology and psychotherapy for an optimal outcome. This case is brought out here for florid presentation and nuances of management. PMID:27212827

  20. 19. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    19. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects September 12, 1907 ORIGINAL DRAWING OF THIRD FLOOR PLAN From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  1. 15. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects May 10, 1907 ORIGINAL DRAWING OF WEST ELEVATION AND DETAILS From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  2. 17. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    17. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects May 3, 1907 ORIGINAL DRAWING OF FIRST FLOOR PLAN From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  3. 14. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects May 10, 1907 ORIGINAL DRAWING OF SOUTH ELEVATION AND DETAILS From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  4. 21. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects September 16, 1907 ORIGINAL DRAWING OF STAIR HALL DETAILS From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  5. 16. Historic American Buildings Survey M. Diggs, Draftsman of Gill ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. Historic American Buildings Survey M. Diggs, Draftsman of Gill and Mead, Architects May 10, 1907 ORIGINAL DRAWING OF NORTH ELEVATION AND DETAILS From the Collection of Mr. Allan Klauber - Melville Klauber House, 3060 Sixth Avenue, San Diego, San Diego County, CA

  6. Myxosporean hyperparasites of gill monogeneans are basal to the Multivalvulida

    PubMed Central

    2011-01-01

    Background Myxosporeans are known from aquatic annelids but parasitism of platyhelminths by myxosporeans has not been widely reported. Hyperparasitism of gill monogeneans by Myxidium giardi has been reported from the European eel and Myxidium-like hyperparasites have also been observed during studies of gill monogeneans from Malaysia and Japan. The present study aimed to collect new hyperparasite material from Malaysia for morphological and molecular descriptions. In addition, PCR screening of host fish was undertaken to determine whether they are also hosts for the myxosporean. Results Heavy myxosporean infections were observed in monogeneans from two out of 14 fish and were detected from a further five fish using specific PCRs and pooled monogenean DNA. Positive DNA isolates were sequenced and were from a single species of myxosporean. Myxospore morphology was consistent with Myxidium with histozoic development in the parenchymal tissues of the monogenean. Simultaneous infections in the fish could not be confirmed microscopically; however, identical myxosporean DNA could be amplified from kidney, spleen and intestinal tract tissues using the specific PCR. Small subunit (SSU) rDNA for the myxosporean was amplified and was found to be most similar (92%) to that of another hyperparasitic myxosporean from a gill monogenean from Japan and to numerous multivalvulidan myxosporeans from the genus Kudoa (89-91%). Phylogenetic analyses placed the hyperparasite sequence basally to clades containing Kudoa, Unicapsula and Sphaerospora. Conclusions The myxosporean infecting the gill monogenean, Diplectanocotyla gracilis, from the Indo-Pacific tarpon, Megalops cyprinoides, is described as a new species, Myxidium incomptavermi, based on a histozoic development in the monogenean host and its phylogenetic placement. We have demonstrated for the first time that a myxosporean hyperparasite of gill monogeneans is detectable in the fish host. However, myxospores could not be isolated

  7. Structure-activity relationships among the ortho-, meta- and para-isomers of phenylenebis (methylene)seleno cyanate (XSC) as inhibitors of 7,12-dimethylbenz(a)anthracene-DNA binding in mammary glands of female CD rats.

    PubMed

    Chae, Y; Upadhyaya, P; Elbayoumy, K

    1997-01-01

    As shown earlier p-XSC inhibits DMBA-induced mammary cancer in female CD rats. This inhibition is due, in part, to inhibition of DMBA-DNA adduct formation in the target organ. We have now utilized the DMBA-DNA binding assay to evaluate the chemopreventive potential of positional isomers of XSC (o-, m- and p-XSC) applied at selenium doses of 5 and 15 ppm; p-XTC, the sulfur analog of p-XSC, was used at an equimolar dose to determine whether selenium is required for the observed inhibitory effect. Selenium and sulfur compounds were administered in a semipurified high-fat diet (23.5% corn oil). Rats were fed for 1 week prior to oral administration of a single dose of [H-3]DMBA (5 mg/rat); animals were sacrificed 24 h later, DNA was isolated from mammary fat pads and levels of total binding were determined. All agents produced a dose-dependent inhibition of DMBA-DNA binding in the mammary tissues. The inhibition at 5, respectively 15 ppm Se in the form of XSC isomers and at 30 mu M, respectively 90 mu M in the form of p-XTC was: o-XSC (27%, 42%); m-XSC (32%, 47%); p-XSC (22%, 29%); and p-XTC (10%, 20%); only inhibition by dietary o-XSC and m-XSC at 15 ppm Se reached statistical significance (p<0.05). Thus, o-XSC and m-XSC equally inhibit DMBA-DNA binding and both are better inhibitors than p-XSC; the latter appears to be slightly more effective than its sulfur analog p-XTC. Clearly, the structure of the selenium-containing compound is a critical factor in determining the extent of inhibition of DMBA-DNA binding. The described short-term in vivo assay may constitute the basis for future selection of chemopreventive agents in the rat mammary tumor model system.

  8. Dynamic features of the selective pressure on the human immunodeficiency virus type 1 (HIV-1) gp120 CD4-binding site in a group of long term non progressor (LTNP) subjects

    PubMed Central

    Canducci, Filippo; Marinozzi, Maria Chiara; Sampaolo, Michela; Berrè, Stefano; Bagnarelli, Patrizia; Degano, Massimo; Gallotta, Giulia; Mazzi, Benedetta; Lemey, Philippe; Burioni, Roberto; Clementi, Massimo

    2009-01-01

    The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection. PMID:19146663

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

    PubMed Central

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

    2013-01-01

    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. PMID:23634281

  10. 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. PMID:23634281

  11. Differential expression of CD8 epitopes amongst porcine CD8-positive functional lymphocyte subsets.

    PubMed Central

    Yang, H; Parkhouse, R M

    1997-01-01

    The swine is a useful model for immunobiological studies as it has a highly heterogeneous lymphocyte pool, containing several subsets not easily accessible in humans and rodents. In particular, the CD8-positive (CD8+) cells contain a variety of lymphocyte subsets, such as alpha beta-T cells, gamma delta-T cells, CD4 CD8 double-positive (DP) cells and natural killer (NK) cells. In order to define these subsets further, we have selected four monoclonal antibodies (mAb) with differential reactivity on CD8+ cells. Thus, mAb CD8.1 (PPT20) bound to CD8hi and CD8lo subpopulations in a similar way to the conventional anti-CD8. The mAb CD8.2 (PPT21), though binding to all of the CD8+ cells, reacted preferably with CD8hi. Two other mAb, CD8.3 (PPT22) and CD8.4 (PPT23), were specific for CD8hi alpha beta-T-cell subpopulation. These results, complemented by immunoprecipitation, co-modulation and enzyme-linked immunosorbent assay experiments, suggest that CD8.1 and CD8.2 react putatively with the CD8 alpha-chain and CD8.3 and CD8.4 with the CD8 beta-chain. Tissue distribution studies revealed that CD8+ thymocytes and peripheral CD8hi alpha beta-T cells expressed both putative CD8 alpha- and beta-chains while peripheral CD4+ CD8+ alpha beta-T cells, CD8lo gamma delta-T cells and NK cells expressed only putative CD8 alpha-chain. Functional studies indicated that the CD8hi alpha beta-T and CD8lo gamma delta-T cells were effector cells in the CD3-redirected cytotoxicity. Images Figure 4 PMID:9370923

  12. Pb and Cd binding to natural freshwater biofilms developed at different pH: the important role of culture pH.

    PubMed

    Hua, Xiuyi; Dong, Deming; Ding, Xiaoou; Yang, Fan; Jiang, Xu; Guo, Zhiyong

    2013-01-01

    The effects of solution pH on adsorption of trace metals to different types of natural aquatic solid materials have been studied extensively, but few studies have been carried out to investigate the effect of pH at which the solid materials were formed on the adsorption. The purpose of present study is to examine this effect of culture pH on metal adsorption to natural freshwater biofilms. The adsorption of Pb and Cd to biofilms which were developed at different culture pH values (ranging from 6.5 to 9.0) was measured at the same adsorption pH value (6.5). The culture pH had considerable effects on both composition and metal adsorption ability of the biofilms. Higher culture pH usually promoted the accumulation of organic material and Fe oxides in the biofilms. The culture pH also affected the quantity and species of algae in the biofilms. The adsorption of Pb and Cd to the biofilms generally increased with the increase of culture pH. This increase was minor at lower pH range and significant at higher pH range and was more remarkable for Cd adsorption than for Pb adsorption. The notable contribution of organic material to the adsorption at higher culture pH values was also observed. The profound impacts of culture pH on adsorption behavior of biofilms mainly resulted from the variation of total contents of the biofilm components and were also affected by the alteration of composition and properties of the components. PMID:22562344

  13. Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae

    PubMed Central

    Yutin, Natalya; Tengs, Torstein; Senkevich, Tania; Koonin, Eugene; Rønning, Hans Petter; Alarcon, Marta; Ylving, Sonja; Lie, Kai-Inge; Saure, Britt; Tran, Linh; Dale, Ole Bendik

    2015-01-01

    ABSTRACT Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures. IMPORTANCE Aquaculture is an increasingly important global

  14. Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

    PubMed

    Molina, H; Brenner, C; Jacobi, S; Gorka, J; Carel, J C; Kinoshita, T; Holers, V M

    1991-07-01

    The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.

  15. 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.

  16. PI3Kδ promotes CD4+ T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1

    PubMed Central

    Garçon, Fabien; Okkenhaug, Klaus

    2016-01-01

    Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice. PMID:26740009

  17. Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy.

    PubMed

    Omichinski, J G; Clore, G M; Sakaguchi, K; Appella, E; Gronenborn, A M

    1991-11-01

    A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein. PMID:1959614

  18. An improved girthometer for studies of gill net selectivity

    USGS Publications Warehouse

    Wydoski, Richard S.; Wolfert, David R.

    1968-01-01

    Gill nets are effective for collecting samples of many fish species. These nets may be highly selective in their catch, depending on the mesh size or sizes used and on the size distribution and body shape of the fish in the population. Early studies related mesh selectivity to length or, in a few instances, to length and weight. Later studies showed that the selectivity of gill nets was related more closely to the girth of the fish than to the length, weight, or a combination of these. The girthometer presented here was designed for rapid and accurate measurement of the fish girth. Both speed and accuracy are important when very large numbers of fish are sampled for mesh-selectivity studies. It is also important that a girthometer be sturdy and rigid enough to provide accurate measurements in spite of rough handling and difficult working conditions.

  19. 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.

  20. CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

    PubMed

    Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

    1990-11-01

    The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. PMID:2146997

  1. CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

    PubMed Central

    Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

    1990-01-01

    The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. Images Fig. 3 Fig. 4 Fig. 5 PMID:2146997

  2. Enhanced habit formation in Gilles de la Tourette syndrome.

    PubMed

    Delorme, Cécile; Salvador, Alexandre; Valabrègue, Romain; Roze, Emmanuel; Palminteri, Stefano; Vidailhet, Marie; de Wit, Sanne; Robbins, Trevor; Hartmann, Andreas; Worbe, Yulia

    2016-02-01

    Tics are sometimes described as voluntary movements performed in an automatic or habitual way. Here, we addressed the question of balance between goal-directed and habitual behavioural control in Gilles de la Tourette syndrome and formally tested the hypothesis of enhanced habit formation in these patients. To this aim, we administered a three-stage instrumental learning paradigm to 17 unmedicated and 17 antipsychotic-medicated patients with Gilles de la Tourette syndrome and matched controls. In the first stage of the task, participants learned stimulus-response-outcome associations. The subsequent outcome devaluation and 'slip-of-action' tests allowed evaluation of the participants' capacity to flexibly adjust their behaviour to changes in action outcome value. In this task, unmedicated patients relied predominantly on habitual, outcome-insensitive behavioural control. Moreover, in these patients, the engagement in habitual responses correlated with more severe tics. Medicated patients performed at an intermediate level between unmedicated patients and controls. Using diffusion tensor imaging on a subset of patients, we also addressed whether the engagement in habitual responding was related to structural connectivity within cortico-striatal networks. We showed that engagement in habitual behaviour in patients with Gilles de la Tourette syndrome correlated with greater structural connectivity within the right motor cortico-striatal network. In unmedicated patients, stronger structural connectivity of the supplementary motor cortex with the sensorimotor putamen predicted more severe tics. Overall, our results indicate enhanced habit formation in unmedicated patients with Gilles de la Tourette syndrome. Aberrant reinforcement signals to the sensorimotor striatum may be fundamental for the formation of stimulus-response associations and may contribute to the habitual behaviour and tics of this syndrome. PMID:26490329

  3. The endogenous opioid system in Gilles de la Tourette syndrome.

    PubMed

    Gillman, M A; Sandyk, R

    1986-04-01

    The various neurotransmitter systems postulated to be involved in the pathogenesis of Gilles de la Tourette syndrome(GTS) are described with special reference to the endogenous opioid system(EOS). Malfunction of the opioid system is proposed as the underlying disturbance in this disease causing secondary dysfunction of the other systems. Furthermore, the various symptoms of the illness are examined also in terms of dysfunction of the endogenous opioid system.

  4. Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.

    PubMed

    Perry, A; Lian, L Y; Scrutton, N S

    2001-02-15

    A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin. PMID

  5. Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.

    PubMed Central

    Perry, A; Lian, L Y; Scrutton, N S

    2001-01-01

    A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin. PMID

  6. Reynolds number effects on gill pumping mechanics in mayfly nymphs

    NASA Astrophysics Data System (ADS)

    Sensenig, Andrew; Shultz, Jeffrey; Kiger, Ken

    2006-11-01

    Mayfly nymphs have an entirely aquatic life stage in which they frequently inhabit stagnant water. Nymphs have the capability to generate a ventilation current to compensate for the low oxygen level of the water by beating two linear arrays of plate-like gills that typically line the lateral edge of the abdomen. The characteristic Reynolds number associated with the gill motion changes with animal size, varying over a span of Re = 5 to 100 depending on age and species. The assumption that the system maintains optimal energetic efficiency leads to the prediction that animals transition from rowing to flapping mechanisms with increasing Re, while possibly utilizing a squeeze mechanism to a greater extent at lower Re. To investigate this hypothesis, we capture the motion of the gills through 3D imaging to investigate the effect of Reynolds number on the stroke patterns. PIV is utilized to assess flow rates and viscous dissipation. The effectiveness of the ventilation mechanism at each size has important consequences for the range of oxygen levels, and hence the habitat range, that can be tolerated by that size.

  7. Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus argenteus from a polluted river in southeast Brazil.

    PubMed

    Procópio, Marcela Santos; Ribeiro, Heder José; Pereira, Luciano Almeida; Oliveira Lopes, Gabriel Augusto; Castro, Antônio Carlos Santana; Rizzo, Elizete; Sato, Yoshimi; Russo, Remo Castro; Corrêa, José Dias

    2014-07-01

    The fish gill is in direct and standing contact with the immediate external environment and, therefore, is highly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two different points in São Francisco river. The first point is located near Três Marias dam, while the second is placed downstream the Abaeté river. Chemical approaches showed the presence of metals contamination in the first point. Thus, the main goal of this study was to investigate the possible toxic effects of these contaminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish were obtained in order to calculate the condition factor as an organismal biomarker. The histological changes in gills and alterations in mucous and rodlet cells occurrence were detected microscopically and evaluated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were also assessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals at the two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cell hyperplasia, lamellar fusion, lamellar edema and inflammatory foci varied according to the point. Additionally, mucous and rodlet cells and MPO and EPO activities showed variability according to the environmental conditions. Furthermore, with exception of lamellar hyperplasia and eosinophil peroxidase activity, all others parameters showed sex-variation responses. At the first point, male fish showed a chronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence of inflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an opposite pattern of response to the same environmental conditions. Therefore, we suggest the use of such biomarkers in future monitoring of aquatic systems, taking into account the sex-variation responses. PMID:24795082

  8. Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus argenteus from a polluted river in southeast Brazil.

    PubMed

    Procópio, Marcela Santos; Ribeiro, Heder José; Pereira, Luciano Almeida; Oliveira Lopes, Gabriel Augusto; Castro, Antônio Carlos Santana; Rizzo, Elizete; Sato, Yoshimi; Russo, Remo Castro; Corrêa, José Dias

    2014-07-01

    The fish gill is in direct and standing contact with the immediate external environment and, therefore, is highly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two different points in São Francisco river. The first point is located near Três Marias dam, while the second is placed downstream the Abaeté river. Chemical approaches showed the presence of metals contamination in the first point. Thus, the main goal of this study was to investigate the possible toxic effects of these contaminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish were obtained in order to calculate the condition factor as an organismal biomarker. The histological changes in gills and alterations in mucous and rodlet cells occurrence were detected microscopically and evaluated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were also assessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals at the two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cell hyperplasia, lamellar fusion, lamellar edema and inflammatory foci varied according to the point. Additionally, mucous and rodlet cells and MPO and EPO activities showed variability according to the environmental conditions. Furthermore, with exception of lamellar hyperplasia and eosinophil peroxidase activity, all others parameters showed sex-variation responses. At the first point, male fish showed a chronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence of inflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an opposite pattern of response to the same environmental conditions. Therefore, we suggest the use of such biomarkers in future monitoring of aquatic systems, taking into account the sex-variation responses.

  9. Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-beta binding in B cells.

    PubMed

    Cruickshank, Mark N; Fenwick, Emily; Karimi, Mahdad; Abraham, Lawrence J; Ulgiati, Daniela

    2009-08-01

    Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

  10. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

    PubMed

    Georgiou, Dimitra K; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M; Wang, Hui; Lagor, William R; Pautler, Robia G; Dirksen, Robert T; Hamilton, Susan L

    2015-09-25

    Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle.

  11. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism*

    PubMed Central

    Georgiou, Dimitra K.; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M.; Wang, Hui; Lagor, William R.; Pautler, Robia G.; Dirksen, Robert T.; Hamilton, Susan L.

    2015-01-01

    Ca2+ permeation and/or binding to the skeletal muscle L-type Ca2+ channel (CaV1.1) facilitates activation of Ca2+/calmodulin kinase type II (CaMKII) and Ca2+ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca2+ binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle. PMID:26245899

  12. Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts*

    PubMed Central

    Lang, Isabell; Fick, Andrea; Schäfer, Viktoria; Giner, Tina; Siegmund, Daniela; Wajant, Harald

    2012-01-01

    The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to “mark” inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules. PMID:22645131

  13. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    PubMed

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference. PMID:27030289

  14. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    PubMed

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference.

  15. 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.

  16. Developmental evidence for serial homology of the vertebrate jaw and gill arch skeleton.

    PubMed

    Gillis, J Andrew; Modrell, Melinda S; Baker, Clare V H

    2013-01-01

    Gegenbaur's classical hypothesis of jaw-gill arch serial homology is widely cited, but remains unsupported by either palaeontological evidence (for example, a series of fossils reflecting the stepwise transformation of a gill arch into a jaw) or developmental genetic data (for example, shared molecular mechanisms underlying segment identity in the mandibular, hyoid and gill arch endoskeletons). Here we show that nested expression of Dlx genes--the 'Dlx code' that specifies upper and lower jaw identity in mammals and teleosts--is a primitive feature of the mandibular, hyoid and gill arches of jawed vertebrates. Using fate-mapping techniques, we demonstrate that the principal dorsal and ventral endoskeletal segments of the jaw, hyoid and gill arches of the skate Leucoraja erinacea derive from molecularly equivalent mesenchymal domains of combinatorial Dlx gene expression. Our data suggest that vertebrate jaw, hyoid and gill arch cartilages are serially homologous, and were primitively patterned dorsoventrally by a common Dlx blueprint.

  17. 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.

  18. Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

    PubMed

    Rivera-Ingraham, Georgina A; Barri, Kiam; Boël, Mélanie; Farcy, Emilie; Charles, Anne-Laure; Geny, Bernard; Lignot, Jehan-Hervé

    2016-01-01

    Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2 weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress.

  19. 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.

  20. Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains.

    PubMed

    Katagiri, Y U; Mori, T; Nakajima, H; Katagiri, C; Taguchi, T; Takeda, T; Kiyokawa, N; Fujimoto, J

    1999-12-01

    Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, N., Horie, H., Takeda, T and Fujimoto, J. (1998) Kidney Int. 53, 1681-1688). To study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction increased by 10 min and returned to the resting level by 30 min after the addition of Stx. Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction.

  1. Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors.

    PubMed

    Olson, Kenneth R; Healy, Michael J; Qin, Zhaohong; Skovgaard, Nini; Vulesevic, Branka; Duff, Douglas W; Whitfield, Nathan L; Yang, Guangdong; Wang, Rui; Perry, Steve F

    2008-08-01

    O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.

  2. [From tics to the Gilles de la Tourette syndrome].

    PubMed

    Kecman-Sugić, M

    1993-01-01

    We report a case whose progressive course and forms of manifestation of generalized tic movements indicate relatively rare Gilles de la Tourette disease. It is characterized by the triad--non-coordinated mobility-dyskinesia, echolalia corporalia, epilepsy and according to the current data from the literature, compulsive behavior, phobias and sometimes episodes of insanity in the advanced stage. The clinical case we report as well as cases from the world literature is rather dubious, asks for prolonged follow-up and has polymorphic symptomatology and necessitates a multidisciplinary therapeutic approach. Beside the clinical features, laboratory findings has also been discussed.

  3. 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.

  4. Lagrangian approach to understanding the origin of the gill-kinematics switch in mayfly nymphs

    NASA Astrophysics Data System (ADS)

    Chabreyrie, R.; Balaras, E.; Abdelaziz, K.; Kiger, K.

    2014-12-01

    The mayfly nymph breathes under water through an oscillating array of plate-shaped tracheal gills. As the nymph grows, the kinematics of these gills change abruptly from rowing to flapping. The classical fluid dynamics approach to consider the mayfly nymph as a pumping device fails in giving clear reasons for this switch. In order to shed some light on this switch between the two distinct kinematics, we analyze the problem under a Lagrangian viewpoint. We consider that a good Lagrangian transport that effectively distributes and stirs water and dissolved oxygen between and around the gills is the main goal of the gill motion. Using this Lagrangian approach, we are able to provide possible reasons behind the observed switch from rowing to flapping. More precisely, we conduct a series of in silico mayfly nymph experiments, where body shape, as well as gill shapes, structures, and kinematics are matched to those from in vivo. In this paper, we show both qualitatively and quantitatively how the change of kinematics enables better attraction, confinement, and stirring of water charged of dissolved oxygen inside the gills area. We reveal the attracting barriers to transport, i.e., attracting Lagrangian coherent structures, that form the transport skeleton between and around the gills. In addition, we quantify how well the fluid particles are stirred inside the gills area, which by extension leads us to conclude that it will increase the proneness of molecules of dissolved oxygen to be close enough to the gills for extraction.

  5. Morphological and biochemical variations in the gills of 12 aquatic air-breathing anabantoid fish.

    PubMed

    Huang, Chun-Yen; Lin, Chung-Ping; Lin, Hui-Chen

    2011-01-01

    All fish species in the Anabantoidei suborder are aquatic air-breathing fish. These species have an accessory air-breathing organ, called the labyrinth organ, in the branchial cavity and can engulf air at the surface of the water to assist in gas exchange. It is therefore necessary to examine the extent of gill modification among anabantoid fish species and the potential trade-offs in their function. The experimental hypothesis that we aimed to test is whether anabantoid fishes have both morphological and functional variations in the gills among different species. We examined the gills of 12 species from three families and nine genera of Anabantoidei. Though the sizes of the fourth gill arch in three species of Trichogaster were reduced significantly, not all anabantoid species had morphological and functional variations in the gills. In these three species, the specific enzyme activity and relative protein abundance of Na(+)/K(+)-ATPase were significantly higher in the anterior gills as compared with the posterior gills and the labyrinth organ. The relative abundance of cytosolic carbonic anhydrase, an indicator of gas exchange, was found to be highest in the labyrinth organ. The phylogenetic distribution of the fourth gill's morphological differentiation suggests that these variations are lineage specific, which may imply a phylogenetic influence on gill morphology in anabantoid species.

  6. 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.

  7. Identification of a novel nonstructural protein, VP9, from white spot syndrome virus: its structure reveals a ferredoxin fold with specific metal binding sites.

    PubMed

    Liu, Yang; Wu, Jinlu; Song, Jianxing; Sivaraman, J; Hew, Choy L

    2006-11-01

    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.

  8. 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.

  9. Redox proteomic analysis of mytilus edulis gills: effects of the pharmaceutical diclofenac on a non-target organism.

    PubMed

    Jaafar, Siti Nur Tahirah; Coelho, Ana Varela; Sheehan, David

    2015-10-01

    Veterinary and human pharmaceuticals are an emerging category of chemical pollutants with potential to cause serious toxicity to non-target organisms. Filter-feeding aquatic organisms such as mussels are especially threatened. In this study, the blue mussel, Mytilus edulis, was exposed to two doses (0.2 mg/L and 1 mg/L) of the anti-inflammatory diclofenac. Effects on the gill, the principal feeding organ of mussels, were investigated. It was noted that, while no effect was evident on gill glutathione transferase or catalase activities, there was a tissue-specific increase in glutathione reductase activity and reduction in total protein thiol groups. Two dimensional electrophoresis was performed and some affected proteins identified by in-gel tryptic digestion and peptide mass fingerprinting. Of these, four unique proteins (caspase 3/7-4, heat-shock cognate protein 70, a predicted enolase-like protein, arginine kinase) were found to be oxidized whilst eight unique proteins (β-tubulin, actin, isocitrate dehydrogenase, arginine kinase, heavy metal-binding HIP, cytosolic malate dehydrogenase, proteasome subunit alpha type 2, Mg: bb02e05 (glyceraldehyde-3-phosphate dehydrogenase) and superoxide dismutase) were found to have altered abundance. In addition, bioinformatic analysis suggested putative identities for six hypothetical proteins which either were oxidized or decreased in abundance. These were; 78 kDa glucose-regulated protein precursor, α-enolase, calreticulin, mitochondrial H + -ATPase, palmitoyl protein thioesterase 1 and initiation factor 5a. It is concluded that diclofenac causes significant oxidative stress to gills and that this affects key structural, metabolic and stress-response proteins. PMID:25833337

  10. 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.

  11. Redox proteomic analysis of mytilus edulis gills: effects of the pharmaceutical diclofenac on a non-target organism.

    PubMed

    Jaafar, Siti Nur Tahirah; Coelho, Ana Varela; Sheehan, David

    2015-10-01

    Veterinary and human pharmaceuticals are an emerging category of chemical pollutants with potential to cause serious toxicity to non-target organisms. Filter-feeding aquatic organisms such as mussels are especially threatened. In this study, the blue mussel, Mytilus edulis, was exposed to two doses (0.2 mg/L and 1 mg/L) of the anti-inflammatory diclofenac. Effects on the gill, the principal feeding organ of mussels, were investigated. It was noted that, while no effect was evident on gill glutathione transferase or catalase activities, there was a tissue-specific increase in glutathione reductase activity and reduction in total protein thiol groups. Two dimensional electrophoresis was performed and some affected proteins identified by in-gel tryptic digestion and peptide mass fingerprinting. Of these, four unique proteins (caspase 3/7-4, heat-shock cognate protein 70, a predicted enolase-like protein, arginine kinase) were found to be oxidized whilst eight unique proteins (β-tubulin, actin, isocitrate dehydrogenase, arginine kinase, heavy metal-binding HIP, cytosolic malate dehydrogenase, proteasome subunit alpha type 2, Mg: bb02e05 (glyceraldehyde-3-phosphate dehydrogenase) and superoxide dismutase) were found to have altered abundance. In addition, bioinformatic analysis suggested putative identities for six hypothetical proteins which either were oxidized or decreased in abundance. These were; 78 kDa glucose-regulated protein precursor, α-enolase, calreticulin, mitochondrial H + -ATPase, palmitoyl protein thioesterase 1 and initiation factor 5a. It is concluded that diclofenac causes significant oxidative stress to gills and that this affects key structural, metabolic and stress-response proteins.

  12. 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. PMID:27335260

  13. Barrier cells in gills of the alligator gar.

    PubMed

    Henderson, V

    1999-10-01

    Studies of barrier cells in fishes are few and have been restricted primarily to salmonids. Moreover, these studies revealed the presence of barrier cells only in hematopoietic and lymphoid organs. The alligator gar, Lepisosteus spatula, possesses similar tissues within the gills which are readily accessible; therefore, they were the organs of choice. An electron microscopical analysis of the gills was undertaken to determine if barrier cells exist and if they possess an ultrastructural design comparable to their counterparts in the brain and lungs of higher vertebrates and hematopoietic/lymphoid tissues in fishes. The present study revealed that barrier cells were found only within non-hematopoietic/lymphoid areas. Barrier cells surrounded endocrine components, nerves, and sinusoids rather than capillary endothelium or hematopoietic/lymphoid tissues. Barrier cells in the alligator gar displayed a complex envelopment more similar to those found in the blood-cerebrospinal fluid, blood-brain, and blood-gas barriers in higher vertebrates than in salmonids. The barrier in the alligator gar consisted of: (1) an endothelium whose cells displayed tight junctions; (2) a basement membrane; and (3) an outer adventitia composed of fibrocytic cells in syncytium. PMID:18627869

  14. Barrier cells in gills of the alligator gar.

    PubMed

    Henderson, V

    1999-10-01

    Studies of barrier cells in fishes are few and have been restricted primarily to salmonids. Moreover, these studies revealed the presence of barrier cells only in hematopoietic and lymphoid organs. The alligator gar, Lepisosteus spatula, possesses similar tissues within the gills which are readily accessible; therefore, they were the organs of choice. An electron microscopical analysis of the gills was undertaken to determine if barrier cells exist and if they possess an ultrastructural design comparable to their counterparts in the brain and lungs of higher vertebrates and hematopoietic/lymphoid tissues in fishes. The present study revealed that barrier cells were found only within non-hematopoietic/lymphoid areas. Barrier cells surrounded endocrine components, nerves, and sinusoids rather than capillary endothelium or hematopoietic/lymphoid tissues. Barrier cells in the alligator gar displayed a complex envelopment more similar to those found in the blood-cerebrospinal fluid, blood-brain, and blood-gas barriers in higher vertebrates than in salmonids. The barrier in the alligator gar consisted of: (1) an endothelium whose cells displayed tight junctions; (2) a basement membrane; and (3) an outer adventitia composed of fibrocytic cells in syncytium.

  15. CD300c is uniquely expressed on CD56bright Natural Killer Cells and differs from CD300a upon ligand recognition

    PubMed Central

    Dimitrova, Milena; Zenarruzabeitia, Olatz; Borrego, Francisco; Simhadri, Venkateswara R.

    2016-01-01

    Paired receptors on NK cells recognize similar ligands with varied strength of binding ability and perform different functions. The CD300 molecules are emerging as novel immune regulators in health and disease due to their interaction with their lipid-nature ligands. Particularly, the paired receptors CD300c and CD300a have been shown to elicit activating and inhibitory capabilities, respectively. In the current study, we seek to investigate the expression and function of CD300c on human NK cells. We demonstrate that IL-2 and IL-15 treatment significantly induce CD300c expression exclusively on CD56bright NK cells. CD300c up-regulation requires STAT5 and its expression is inhibited by IL-4. Consistently, IL-2 secreted from activated CD4+ T cells specifically induces the expression of CD300c on CD56bright NK cells. Crosslinking CD300c with a specific antibody enhances the proficiency of CD56bright NK cells to degranulate and induce chemokine and cytokine secretion. We also show the differential binding of CD300a and CD300c to their ligands phosphatidylethanolamine (PE) and phosphatidylserine (PS) and their differential ability to affect CD56bright NK cell functions. Our results provide an insight into the novel set of paired receptors CD300a and CD300c that are distinctively expressed on CD56bright NK cells with varied effector functions. PMID:27040328

  16. External gills and adaptive embryo behavior facilitate synchronous development and hatching plasticity under respiratory constraint.

    PubMed

    Rogge, Jessica R; Warkentin, Karen M

    2008-11-01

    Plasticity in hatching timing allows embryos to balance egg- and larval-stage risks, and depends on the ability of hatching-competent embryos to continue developing in the egg. Hypoxia can slow development, kill embryos and induce premature hatching. For terrestrial eggs of red-eyed treefrogs, the embryonic period can extend approximately 50% longer than development to hatching competence, and development is synchronous across perivitelline oxygen levels (PO2) ranging from 0.5-16.5 kPa. Embryos maintain large external gills until hatching, then gills regress rapidly. We assessed the respiratory value of external gills using gill manipulations and closed-system respirometry. Embryos without external gills were oxygen limited in air and hatched at an external PO2 of 17 kPa, whereas embryos with gills regulated their metabolism and remained in the egg at substantially lower PO2. By contrast, tadpoles gained no respiratory benefit from external gills. We videotaped behavior and manipulated embryos to test if they position gills near the air-exposed portion of the egg surface, where PO2 is highest. Active embryos remained stationary for minutes in gills-at-surface positions. After manipulations and spontaneous movements that positioned gills in the O2-poor region of the egg, however, they returned their gills to the air-exposed surface within seconds. Even neural tube stage embryos, capable only of ciliary rotation, positioned their developing head in the region of highest PO2. Such behavior may be critical both to delay hatching after hatching competence and to obtain sufficient oxygen for normal, synchronous development at earlier stages.

  17. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

    PubMed Central

    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

    2015-01-01

    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. PMID:26556861

  18. 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.

  19. Intraparticulate speciation analysis of soft nanoparticulate metal complexes. The impact of electric condensation on the binding of Cd²⁺/Pb²⁺/Cu²⁺ by humic acids.

    PubMed

    Town, Raewyn M; van Leeuwen, Herman P

    2016-04-21

    In aqueous dispersions of soft, charged nanoparticles, the physicochemical conditions prevailing within the particle body generally differ substantially from those in the bulk medium. Accordingly it is necessary to define intrinsic descriptors that appropriately reflect the chemical speciation inside the particle's microenvironment. Herein the speciation of divalent metal ions within the body of negatively charged soft nanoparticulate complexants is elaborated for the example case of humic acid association with Cd(ii), Pb(ii) and Cu(ii). The electrostatic effects are described by a two-state model that accounts for counterion condensation in the intraparticulate double layer shell at the particle/medium interface and Donnan partitioning within the bulk of the particle body. Inner-sphere complex formation is defined by an intrinsic binding constant expressed in terms of local reactant concentrations as controlled by the pertinent electrostatic conditions. For the high particle charge density case (Debye length smaller than charged site separation), three distinct intraparticulate metal species are identified, namely free hydrated ions, electrostatically condensed ions, and inner-sphere metal-humic complexes. For all metal ions studied, the electrostatic contribution to the association of the metal ion with the oppositely charged particle is found to account for a substantial fraction of the total metal bound. PMID:27004844

  20. 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

  1. CD Recorders.

    ERIC Educational Resources Information Center

    Falk, Howard

    1998-01-01

    Discussion of CD (compact disc) recorders describes recording applications, including storing large graphic files, creating audio CDs, and storing material downloaded from the Internet; backing up files; lifespan; CD recording formats; continuous recording; recording software; recorder media; vulnerability of CDs; basic computer requirements; and…

  2. 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…

  3. 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...

  4. Training Researchers in Cultural Psychiatry: The McGill-CIHR Strategic Training Program

    ERIC Educational Resources Information Center

    Kirmayer, Laurence J.; Rousseau, Cecile; Corin, Ellen; Groleau, Danielle

    2008-01-01

    Objectives: The authors aim to summarize the pedagogical approaches and curriculum used in the training of researchers in cultural psychiatry at the Division of Social and Transcultural Psychiatry at McGill University. Method: We reviewed available published and unpublished reports on the history and development of the McGill cultural psychiatry…

  5. Mating rendezvous in monogenean gill parasites of the humbug Dascyllus aruanus (Pisces: Pomacentridae).

    PubMed

    Lo, C M

    1999-12-01

    The gills of the humbug, Dascyllus aruanus (Pomacentridae), were infected by a monogenean genus Haliotrema at a high prevalence (83%) but with a low mean intensity (5.6 worms/fish). All the gill arches of 365 fish, caught on the fringing reef of Moorea Island (French Polynesia), were examined for parasites. Each hemibranch was divided into 12 subequal sections. Monogeneans showing microhabitat overlap were defined as couples. Hosts with low intensity of infection (fewer than 5 monogeneans per gill) were selected and couples were recorded. Among the 37 hosts harboring 2 worms on their gills, 18 fish were infected with these 2 monogeneans on the same gill side of the body; 50% (n = 9) of these harbored monogeneans within the same gill arch and 55% (n = 5) of these last fish showed individual parasites within the same section of the gill. In the case of hosts with few monogeneans (3 and 4 individuals; n = 37) on the same arch, more than 40% (n = 16) harbored worms in couples. There may be some chemical communication that allowed these monogeneans to migrate toward each other and thus enhance mating success. Mating rendezvous appears to be a more important factor than site location for these gill monogeneans.

  6. 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. PMID:27095494

  7. 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.

  8. Delayed experience of volition in Gilles de la Tourette syndrome.

    PubMed

    Moretto, Giovanna; Schwingenschuh, Petra; Katschnig, Petra; Bhatia, Kailash P; Haggard, Patrick

    2011-12-01

    Gilles de la Tourette's syndrome (TS) is a neuropsychiatric movement disorder characterised by the presence of multiple tics. Tics have an unusual, intermediate status between voluntary and involuntary movements. This ambiguity might involve not just a disorder of movement generation but also an abnormality of voluntary experience. Here the experience of voluntary movements in adult patients with TS is investigated and compared with healthy controls. A group of adult TS patients and age matched control participants estimated the time of conscious intention to perform a simple keypress movement and movement onset. Patients with TS showed a delayed experience of intention relative to controls whereas estimates of the actual movement onset were similar for patients and controls. These data suggest an abnormal experience of volition in patients with TS. Delayed volition could either be an additional intrinsic feature of the syndrome or it could reflect a cognitive strategy to limit motor excitability, and thus tic generation, during voluntary action.

  9. The psychopathological spectrum of Gilles de la Tourette syndrome.

    PubMed

    Cavanna, Andrea E; Rickards, Hugh

    2013-07-01

    Gilles de la Tourette syndrome (GTS) holds a unique status as quintessentially neuropsychiatric condition at the interface between neurology (movement disorder) and psychiatry (behavioural condition). This is a reflection of the common observation that the vast majority of patients present with behavioural problems in association with the motor and vocal tics which define GTS. The present article focuses on the relationship between GTS and obsessive-compulsive disorder (OCD), attention-deficit and hyperactivity disorder (ADHD), affective disorders (both major depression and bipolar affective disorder), and personality disorders. Over the last decade, converging lines of research have pointed towards the concept of a 'GTS spectrum', encompassing motor phenomena and behavioural symptoms, with important implications for the clinical management of patients.

  10. Gill cell culture systems as models for aquatic environmental monitoring.

    PubMed

    Bury, Nic R; Schnell, Sabine; Hogstrand, Christer

    2014-03-01

    A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples.

  11. Laser-induced spreading arrest of Mytilus gill cilia

    PubMed Central

    1975-01-01

    Using a "slit camera" recording technique, we have examined the effects of local laser irradiation of cilia of the gill epithelium of Mytilus edulis. The laser produces a lesion which interrupts epithelial integrity. In artificial sea water that contains high K+ or is effectively Ca++ free, metachronism of the lateral cilia continues to either side of the lesion with only minor perturbations in frequency synchronization and wave velocity, such as would be expected if metachronal wave coordination is mechanical. However, in normal sea water and other appropriate ionic conditions (i.e., where Ca++ concentration is elevated), in addition to local damage, the laser induces distinct arrest responses of the lateral cilia. Arrest is not mechanically coordinated, since cilia stop in sequence depending on stroke position as well as distance from the lesion. The velocity of arrest under standard conditions is about 3 mm/s, several orders of magnitude faster than spreading velocities associated with diffusion of materials from the injured region. Two responses can be distinguished on the basis of the kinetics of recovery of the arrested regions. These are (a) a nondecremental response that resembles spontaneous ciliary stoppage in the gills, and (b) a decremental response, where arrest nearer the stimulus point is much longer lasting. The slower recovery is often periodic, with a step size approximating lateral cell length. Arrest responses with altered kinetics also occur in laterofrontal cilia. The responses of Mytilus lateral cilia resemble the spreading ciliary arrest seen in Elliptio and arrest induced by electrical and other stimuli, and the decremental response may depend upon electrotonic spread of potential change produced at the stimulus site. If this were coupled to transient changes in Ca++ permeability of the cell membrane, a local rise in Ca++ concentration might inhibit ciliary beat at a sensitive point in the stroke cycle to produce the observed arrest. PMID

  12. Laser-induced spreading arrest of Mytilus gill cilia.

    PubMed

    Motokawa, T; Satir, P

    1975-08-01

    Using a "slit camera" recording technique, we have examined the effects of local laser irradiation of cilia of the gill epithelium of Mytilus edulis. The laser produces a lesion which interrupts epithelial integrity. In artificial sea water that contains high K+ or is effectively Ca++ free, metachronism of the lateral cilia continues to either side of the lesion with only minor perturbations in frequency synchronization and wave velocity, such as would be expected if metachronal wave coordination is mechanical. However, in normal sea water and other appropriate ionic conditions (i.e., where Ca++ concentration is elevated), in addition to local damage, the laser induces distinct arrest responses of the lateral cilia. Arrest is not mechanically coordinated, since cilia stop in sequence depending on stroke position as well as distance from the lesion. The velocity of arrest under standard conditions is about 3 mm/s, several orders of magnitude faster than spreading velocities associated with diffusion of materials from the injured region. Two responses can be distinguished on the basis of the kinetics of recovery of the arrested regions. These are (a) a nondecremental response that resembles spontaneous ciliary stoppage in the gills, and (b) a decremental response, where arrest nearer the stimulus point is much longer lasting. The slower recovery is often periodic, with a step size approximating lateral cell length. Arrest responses with altered kinetics also occur in laterofrontal cilia. The responses of Mytilus lateral cilia resemble the spreading ciliary arrest seen in Elliptio and arrest induced by electrical and other stimuli, and the decremental response may depend upon electrotonic spread of potential change produced at the stimulus site. If this were coupled to transient changes in Ca++ permeability of the cell membrane, a local rise in Ca++ concentration might inhibit ciliary beat at a sensitive point in the stroke cycle to produce the observed arrest.

  13. Bioaccumulation of hydrophobic xenobiotics via gill and gut

    SciTech Connect

    Qiao, P.; Farrell, A.P.

    1995-12-31

    The authors demonstrated that the uptake of three hydrophobic chemicals, 1,2,4-trichlorobenzene (TCB), 1,2,3,4,5-pentachlorobenzene (PeCB), and 2,2{prime},4,4{prime},6,6{prime}-hexachlorobiphenyl (HCB), from water laden with sediments from the Fraser River was just as rapid and to at least the same level in juvenile rainbow trout with pharynx blocked as compared with control, unfed fish. Furthermore, a chemical mass balance analysis revealed that the quantity of PECB and HCB dissolved in water could not account by itself for the quantity of these compounds that had appeared in the fish after 6 days of exposure. Thus, the authors conclude that hydrophobic chemicals such as these with log K{sub ow} values of 5--7 are bioavailable for gill uptake from water laden with sediments from the Fraser River. In a separate experiment, the appearance of TCB, PeCB and HCB in fish eating contaminated food at 2% body mass/day was measured over 12 days. Although the chemical concentrations in the food were 5,000 to 65,000 times higher than the concentrations in the water plus suspended sediments of the earlier experiment, the concentrations of xenobiotics in fish tissues were not significantly higher than in the unfed fish of the earlier experiment. The authors conclude that the bioavailability via the gill of high K{sub ow} compounds from Fraser River sediments is very significant in juvenile rainbow trout. Data also showed that metabolism of TCB, but not PeCB and HCB, was significant.

  14. Measurement and analysis of equilibrium binding titrations: A beginner's guide.

    PubMed

    Beckett, Dorothy

    2011-01-01

    Binding events are central to biology. Simple binding of a substrate to an enzyme initiates catalysis. Formation of protein:protein complexes is integral to signal transduction. Binding of multiple proteins to the ribosomal ribonucleic acid (rRNA) results in ribosome assembly. Consequently, elucidation of mechanisms of biological processes requires binding measurements. Such measurements reveal, among other things, the relevant concentrations required for binding partners to form a complex and are indispensible to understanding the relationship between structure and biological function. This article is intended to serve as a primer for biologists who are contemplating performing binding studies. The focus is on practical aspects of design and analysis of binding measurements for a simple process. The information that one can extract from such measurements is also addressed. Theoretical background on binding for both simple and complex systems can be found in many textbooks and monographs including those by Hammes [Hammes, G. G. (2000). Thermodynamics and Kinetics for the Biological Sciences. Wiley, New York, NY], Weber [Weber, G. (1992). Protein Interactions. Chapman and Hall, New York, NY], and Wyman and Gill [Wyman, J. and Gill, S. J. (1990). Binding and Linkage. University Science Books, Mill Valley, CA]. While the first reference is excellent for beginners, the latter two, in addition to discussion of simple binding, contain theoretical background for complex binding processes.

  15. Environmental and developmental effects on external gill loss in the red-eyed tree frog, Agalychnis callidryas.

    PubMed

    Warkentin, K M

    2000-01-01

    I examined the effects of development, hatching, and oxygen availability on external gill loss in red-eyed tree frogs, Agalychnis callidryas. Under natural conditions, the arboreal embryos maintained large external gills until hatching, which occurred from 5-8 d after oviposition. At hatching, when tadpoles entered the water, external gills began to regress. In older hatchlings this process was extremely rapid. Gill circulation was lost on average within 16 min and sometimes within 5 min. Gills often regressed completely in under 2 h. Younger hatchlings reduced gill circulation, shortened and adducted their gills, then resumed normal circulation for some time after hatching; half had completely lost external gills within 24 h. Experimentally increasing the area of egg surface exposed to the air induced loss of external gills in unhatched embryos. Older hatchlings in hypoxic water without access to air maintained their external gills. This suggests that loss of external gills is a response to increased oxygen availability, rather than a response to hatching per se. Extended maintenance of external gills by large, late-hatching embryos may facilitate continued rapid development in closely packed eggs. PMID:11073790

  16. 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.

  17. 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

  18. 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

  19. Gill erosion and herpesvirus in Crassostrea gigas cultured in Baja California, Mexico.

    PubMed

    Vásquez-Yeomans, Rebeca; García-Ortega, Mauricio; Cáceres-Martínez, Jorge

    2010-03-01

    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.

  20. 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.

  1. Structural Changes in Gill DNA Reveal the Effects of Contaminants on Puget Sound Fish

    SciTech Connect

    Malins, Donald C.; Stegeman, John J.; Anderson, Jack W.; Johnson, Paul M.; Gold, Jordan; Anderson, Katie M.

    2004-04-01

    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 markets 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 IA (CYPIA) 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-0 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 recession 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.

  2. Hatching timing, oxygen availability, and external gill regression in the tree frog, Agalychnis callidryas.

    PubMed

    Warkentin, Karen M

    2002-01-01

    The physiological role of the embryonic external gills in anurans is equivocal. In some species, diffusion alone is clearly sufficient to supply oxygen throughout the embryonic period. In others, morphological elaboration and environmental regulation of the external gills suggest functional importance. Since oxygen stress is a common trigger of hatching, I examined the relationships among hatching timing, oxygen stress, and external gill loss. I worked with the red-eyed tree frog, Agalychnis callidryas, a species with arboreal eggs and aquatic tadpoles in which gill regression is associated with hatching, and hatching timing affects posthatching survival with aquatic predators. Both exposure to a hypoxic gas mixture and submergence in water, a natural context in which hypoxic stress can occur, induced early hatching. Exposure to hyperoxic gas mixtures induced regression of external gills, and subsequent exposure to air induced early hatching. Prostaglandin-induced external gill regression also induced hatching, and this effect was partially ameliorated by exposure to hyperoxic gas. Together, these results suggest that external gills enhance the oxygen uptake of embryos and are necessary to extend embryonic development past the onset of hatching competence. PMID:12024291

  3. Trout gill cells in primary culture on solid and permeable supports.

    PubMed

    Leguen, I; Cauty, C; Odjo, N; Corlu, A; Prunet, P

    2007-12-01

    Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.

  4. 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.

  5. Functional morphology of gill ventilation of the goosefish, Lophius americanus (Lophiiformes: Lophiidae).

    PubMed

    Farina, Stacy C; Bemis, William E

    2016-06-01

    The goosefish, Lophius americanus, is a dorso-ventrally compressed marine fish that spends most of its life sitting on the substrate waiting to ambush prey. Species in the genus Lophius have some of the slowest ventilatory cycles recorded in fishes, with a typical cycle lasting more than 90s. They have a large gill chamber, supported by long branchiostegal rays and ending in a siphon-like gill opening positioned underneath and behind the base of the pectoral fin. Our goals were to characterize the kinematics of gill ventilation in L. americanus relative to those of more typical ray-finned fishes, address previous assertions about ventilation in this genus, and describe the anatomy of the gill opening. We found that phase 1 of ventilation (during which both the buccal and gill chamber are expanding) is greatly increased in duration relative to that of typical ray-finned fishes (ranging from 62 to 127s), and during this phase, the branchiostegal rays are slowly expanding. This slow expansion is almost visually imperceptible, especially from a dorsal view. Despite this unusually long phase 1, the pattern of skeletal movements follows that of a typical actinopterygian, refuting previous assertions that Lophius does not use its jaws, suspensorium, and operculum during ventilation. When individuals were disturbed from the sediment, they tended to breathe more rapidly by decreasing the duration of phase 1 (to 18-30s). Dissections of the gill opening revealed a previously undocumented dorsal extension of the adductor hyohyoideus muscle, which passes from between the branchiostegal rays, through the ventro-medial wall of the gill opening, and to the dorsal midline of the body. This morphology of the adductor hyohyoideus shares similarities with that of many Tetraodontiformes, and we suggest that it may be a synapomorphy for Lophiiformes+Tetraodontiformes. The specialized anatomy and function of the gill chamber of Lophius represents extreme modifications that provide

  6. Members of adenovirus species B utilize CD80 and CD86 as cellular attachment receptors

    PubMed Central

    Short, Joshua J.; Vasu, Chenthamarakshan; Holterman, Mark J.; Curiel, David T.; Pereboev, Alexander

    2007-01-01

    Alternate serotypes of adenovirus (Ad), including Ads of species B, are being explored to circumvent the disadvantages of Ad serotype 5 gene delivery vectors. Whereas the majority of human Ads utilize the Coxsackievirus and adenovirus receptor (CAR), none of the Ad species B use CAR. Ad species B is further divided into two subspecies, B1 and B2, and utilizes at least two classes of receptors: common Ad species B receptors and B2 specific receptors. CD46 has been implicated as a B2-specific receptor. Ad serotype 3 (Ad3), a member of B1, utilizes CD80 and CD86 as cellular attachment receptors. The receptor-interacting Ad fiber-knob domain is highly homologous among species B Ads. We hypothesized that other members of Ad species B may utilize CD80 and CD86 as cellular attachment receptors. All tested species B members showed specific binding to cells expressing CD80 and CD86, and the Ad fiber-knob domain from both B1 and B2 Ad efficiently blocked CD80- and CD86-mediated infection of Ad3 vectors. Members of both B1 and B2 demonstrated CD80- and CD86-specific infection of CHO cells expressing CD80 and CD86. Therefore, all of the members of Ad species B utilize CD80 and CD86 for infection of cells. PMID:16920215

  7. THE CELL JUNCTION IN A LAMELLIBRANCH GILL CILIATED EPITHELIUM

    PubMed Central

    Satir, P.; Gilula, N. B.

    1970-01-01

    The junctional complex in the gill epithelium of the freshwater mussel (Elliptio complanatus) consists of an intermediary junction followed by a 2–3 µ long septate junction. Homologous and heterologous cell pairs are connected by this junction. After fixation with 1% OsO4 containing 1% potassium pyroantimonate, electron microscopy of the gill reveals deposits of electron-opaque precipitate, specifically and consistently localized along cellular membranes. In both junctional and nonjunctional membrane regions, the precipitate usefully outlines the convolutions without obliterating the 150 A intercellular space, which suggests the rarity or absence of either vertebrate-type gap or tight junctions along the entire cell border. The precipitate appears on the cytoplasmic side of the limiting unit membranes of frontal (F), laterofrontal (LF), intermediate (I), lateral (L), and postlateral (PL) cells. The membrane surfaces of certain vesicles of the smooth endoplasmic reticulum, of multivesicular bodies, and of mitochondrial cristae contain precipitate, as does the nucleolus. In other portions of the cell, precipitate is largely absent. The amount of over-all deposition is variable and depends on the treatment of the tissue prior to fixation. Deposition is usually enhanced by pretreatment with 40 mM NaCl as opposed to 40 mM KCl, which suggests that the precipitate is in part sodium pyroantimonate. Treatment with 0.2 mM ouabain does not enhance deposition. Regional differentiation of cell membranes with respect to their ability to precipitate pyroantimonate is found in at least three instances: (a) between the ciliary membranes and other portions of the cell membrane: the precipitate terminates abruptly at the ciliary base, (b) between the LF and I cell borders: the precipitate is asymmetric, favoring the LF side of the junction, and (c) between the septate junctional membrane and adjacent membrane: the precipitate occurs periodically throughout the septate junction

  8. 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

  9. Cadmium affects the mitochondrial viability and the acid soluble thiols concentration in liver, kidney, heart and gills of Ancistrus brevifilis (Eigenmann, 1920).

    PubMed

    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

  10. Altered intrahemispheric structural connectivity in Gilles de la Tourette syndrome.

    PubMed

    Cheng, Bastian; Braass, Hanna; Ganos, Christos; Treszl, Andras; Biermann-Ruben, Katja; Hummel, Friedhelm C; Müller-Vahl, Kirsten; Schnitzler, Alfons; Gerloff, Christian; Münchau, Alexander; Thomalla, Götz

    2014-01-01

    Gilles de la Tourette syndrome (GTS) is a common developmental neuropsychiatric disorder characterized by tics and frequent psychiatric comorbidities, often causing significant disability. Tic generation has been linked to disturbed networks of brain areas involved in planning, controlling and execution of actions, particularly structural and functional disorders in the striatum and cortico-striato-thalamo-cortical loops. We therefore applied structural diffusion tensor imaging (DTI) to characterize changes in intrahemispheric white matter connectivity in cortico-subcortical circuits engaged in motor control in 15 GTS patients without psychiatric comorbidities. White matter connectivity was analyzed by probabilistic fiber tractography between 12 predefined cortical and subcortical regions of interest. Connectivity values were combined with measures of clinical severity rated by the Yale Global Tic Severity Scale (YGTSS). GTS patients showed widespread structural connectivity deficits. Lower connectivity values were found specifically in tracts connecting the supplementary motor areas (SMA) with basal ganglia (pre-SMA-putamen, SMA-putamen) and in frontal cortico-cortical circuits. There was an overall trend towards negative correlations between structural connectivity in these tracts and YGTSS scores. Structural connectivity of frontal brain networks involved in planning, controlling and executing actions is reduced in adult GTS patients which is associated with tic severity. These findings are in line with the concept of GTS as a neurodevelopmental disorder of brain immaturity.

  11. Amoebic gill infection in coho salmon Oncorhynchus kisutch farmed in Korea.

    PubMed

    Kim, Wi-Sik; Kong, Kyoung-Hui; Kim, Jong-Oh; Oh, Myung-Joo

    2016-08-31

    About 70% mortality occurred in cultured coho salmon Oncorhynchus kisutch at a marine farm in the South Sea of Korea in 2014. Diseased fish showed greyish or pale patches on the gills, with no internal signs of disease. No bacteria or viruses were isolated from diseased fish, but numerous amoebae were found on the gills. Histopathological examinations revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae had a 630 bp partial 18S rRNA gene fragment specific to Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that this Korean amoeba belonged to the N. perurans group. This is the first report of N. perurans infection in Korea. PMID:27596862

  12. 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.

  13. 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)

  14. The perfused fish gill preparation in studies of the bioavailability of chemicals

    SciTech Connect

    Paert, P. )

    1990-02-01

    The bioavailability of chemicals has received considerable attention in aquatic toxicology, and it is generally agreed that information about bioavailability is lacking in many situations. Here an experimental approach that can be used to assess the bioavailability of chemicals to fish is presented. The gills are the primary uptake site of dissolved chemicals in fish. A perfused gill preparation from rainbow trout is described. The preparation allows direct measurements of the absorption rates of chemicals across the gill epithelium. The use of the preparation in bioavailability studies is exemplified by a study of cadmium availability in different water qualities, by the pH-dependent absorption of hexavalent chromium and by a study on the pH-dependent absorption of halogenated phenols across the gills.

  15. Ultrastructural effects on gill tissues induced in red tilapia Oreochromis sp. by a waterborne lead exposure.

    PubMed

    Aldoghachi, Mohammed A; Azirun, Mohd Sofian; Yusoff, Ismail; Ashraf, Muhammad Aqeel

    2016-09-01

    Experiments on hybrid red tilapia Oreochromis sp. were conducted to assess histopathological effects induced in gill tissues of 96 h exposure to waterborne lead (5.5 mg/L). These tissues were investigated by light and scanning electron microscopy. Results showed that structural design of gill tissues was noticeably disrupted. Major symptoms were changes of epithelial cells, fusion in adjacent secondary lamellae, hypertrophy and hyperplasia of chloride cells and coagulate necrosis in pavement cells with disappearance of its microridges. Electron microscopic X-ray microanalysis of fish gills exposed to sublethal lead revealed that lead accumulated on the surface of the gill lamella. This study confirmed that lead exposure incited a difference of histological impairment in fish, supporting environmental watch over aquatic systems when polluted by lead. PMID:27579014

  16. 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

  17. The fish gill: site of action and model for toxic effects of environmental pollutants.

    PubMed

    Evans, D H

    1987-04-01

    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.

  18. Fish gill: site of action and model for toxic effects of environmental pollutants

    SciTech Connect

    Evans, D.H.

    1987-04-01

    The gill epithelium is the site of gas exchange, ionic regulation, acid-base balance, 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 underlie 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.

  19. Molecular dissection of the CD2-CD58 counter-receptor interface identifies CD2 Tyr86 and CD58 Lys34 residues as the functional "hot spot".

    PubMed

    Kim, M; Sun, Z Y; Byron, O; Campbell, G; Wagner, G; Wang, J; Reinherz, E L

    2001-09-28

    The heterophilic CD2-CD58 adhesion interface contains interdigitating residues that impart high specificity and rapid binding kinetics. To define the hot spot of this counter-receptor interaction, we characterized CD2 adhesion domain variants harboring a single mutation of the central Tyr86 or of each amino acid residue forming a salt link/hydrogen bond. Alanine mutations at D31, D32 and K34 on the C strand and K43 and R48 on the C' strand reduce affinity for CD58 by 47-127-fold as measured by isothermal titration calorimetry. The Y86A mutant reduces affinity by approximately 1000-fold, whereas Y86F is virtually without effect, underscoring the importance of the phenyl ring rather than the hydroxyl moiety. The CD2-CD58 crystal structure offers a detailed view of this key functional epitope: CD2 D31 and D32 orient the side-chain of CD58 K34 such that CD2 Y86 makes hydrophobic contact with the extended aliphatic component of CD58 K34 between CD2 Y86 and CD58 F46. The elucidation of this hot spot provides a new target for rational design of immunosuppressive compounds and suggests a general approach for other receptors. PMID:11575926

  20. 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.

  1. 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.

  2. 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.

  3. Pathomorphological changes in gills of fish fingerlings (Cirrhina mrigala) by linear alkyl benzene sulfonate

    SciTech Connect

    Misra, V.; Lal, H.; Chawla, G.; Viswanathan, P.N.

    1985-12-01

    Fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of LC50) concentration to detergents (linear alkyl benzene sulfonate) showed marked behavioral changes and distorted appearance of primary and secondary lamellae along with damage to gill epithelium under scanning electron microscopy at various magnifications. Mucosal cells of gills were found to secrete mucus showing primary reactions for membrane damage leading to dysfunction in respiration and osmoregulation.

  4. 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

  5. Respiratory toxicity of cyanobacterial aphantoxins from Aphanizomenon flos-aquae DC-1 in the zebrafish gill.

    PubMed

    Zhang, De Lu; Liu, Si Yi; Zhang, Jing; Zhang, Jian Kun; Hu, Chun Xiang; Liu, Yong Ding

    2016-07-01

    Aphantoxins from Aphanizomenon flos-aquae are frequently identified in eutrophic waterbodies worldwide. These toxins severely endanger environmental safety and human health due to the production of paralytic shellfish poisons (PSPs). Although the molecular mechanisms of aphantoxin neurotoxicity have been studied, many questions remain to be resolved such as in vivo alterations in branchial histology and neurotransmitter inactivation induced by these neurotoxins. Aphantoxins extracted from a naturally isolated strain of A. flos-aquae DC-1 were determined by high performance liquid chromatography. The basic components of the isolated aphantoxins identified were gonyautoxin 1 (GTX1), gonyautoxin 5 (GTX5), and neosaxitoxin (neoSTX), which comprised 34.04, 21.28, and 12.77% of the total, respectively. Zebrafish (Danio rerio) was administrated 5.3 or 7.61mg STX equivalents (eq)/kg (low and high doses, respectively) of the A. flos-aquae DC-1 aphantoxins by intraperitoneal injection. Histological alterations and changes in neurotransmitter inactivation in the gills of zebrafish were investigated for 24h following exposure. Aphantoxin exposure significantly increased the activities of gill alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and resulted in histological alterations in the gills during the first 12h of exposure, indicating the induction of functional and structural damage. Gill acetylcholinesterase (AChE) and monoamine oxidase (MAO) activities were inhibited significantly, suggesting an alteration of neurotransmitter inactivation in zebrafish gills. The observed alterations in gill structure and function followed a time- and dose-dependent pattern. The results demonstrate that aphantoxins or PSPs lead to structural damage and altered function in the gills of zebrafish, including changes in histological structure and increases in the activities of AST and ALT. The inhibition of the activities of AChE and MAO suggest that aphantoxins or PSPs

  6. 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.

  7. Pathomorphological changes in gills of fish fingerlings (Cirrhina mrigala) by linear alkyl benzene sulfonate.

    PubMed

    Misra, V; Lal, H; Chawla, G; Viswanathan, P N

    1985-12-01

    Fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of LC50) concentration to detergents (linear alkyl benzene sulfonate) showed marked behavioral changes and distorted appearance of primary and secondary lamellae along with damage to gill epithelium under scanning electron microscopy at various magnifications. Mucosal cells of gills were found to secrete mucus showing primary reactions for membrane damage leading to dysfunction in respiration and osmoregulation.

  8. CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL.

    PubMed

    Lanier, L L; O'Fallon, S; Somoza, C; Phillips, J H; Linsley, P S; Okumura, K; Ito, D; Azuma, M

    1995-01-01

    Signals initiated through both the TCR complex and CD28 are required for optimal activation of T lymphocytes. Recently, it has been demonstrated that CD28 interacts with two different ligands, designated CD80 (B7/B7-1) and CD86 (B70/B7-2). We have produced stable transfectants that express CD80, CD86, or both ligands and have examined their ability to costimulate T cell proliferation, cytokine production, and the generation of CTL. When we used small, resting human peripheral blood T cells as responders, both CD80 and CD86 transfectants efficiently costimulated anti-CD3 mAb-induced proliferation and the secretion of IL-2 and IFN-gamma. Additionally, both CD80 and CD86 transfectants were able to generate functional CTL. The magnitude and kinetics of these responses were similar, which indicates that both ligands provide efficient costimulatory signals. Because many APCs coexpress both CD80 and CD86, we compared the ability of anti-CD80 and anti-CD86 mAbs to inhibit allogeneic MLR stimulated with B lymphoblastoid cell lines and showed that it is necessary to inhibit interactions with both ligands to optimally block CD28-dependent proliferation. Given the limited homology of CD80 and CD86, it was surprising that the binding of CD28-Ig fusion protein to CD80 and that to CD86 transfectants were essentially indistinguishable. Binding of CTLA-4-Ig fusion protein to both transfectants also was quite similar, but was of higher affinity than CD28-Ig binding. Results from these studies indicate that both CD80 and CD86 are potent and similar costimulators of T lymphocytes. Therefore, the role of CD80 and CD86 in an immune response may be determined primarily by their differential expression on APC.

  9. Prevalence, site and tissue preference of myxozoan parasites infecting gills of cultured fish in Punjab (India).

    PubMed

    Kaur, Harpreet; Katoch, Anu

    2016-02-25

    Native carp species cultured in Indian farms in Punjab (catla Catla catla, rohu Labeo rohita, mrigal Cirrhinus mrigala, exotic carps such as silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella, common carp Cyprinus carpio and a catfish Sperata seenghala) were examined for the presence of myxozoan parasites infecting gills. Firstly, the gills were examined under a zoom-stereomicroscope for the presence of plasmodia. The number of plasmodia per gill was counted to determine the index for the intensity of infection. Infected tissues were processed for histology, and 3-4 µm sections of infected gills were stained with haematoxylin & eosin and Luna's method. A total of 19 species of myxosporean were found infecting various cell types in the gills. Of these, 14 species belonged to the genus Myxobolus, 3 species to the genus Thelohanellus and 2 species to the genus Henneguya. Species belonging to the genus Myxobolus formed the interlamellar and intralamellar vascular (LV) type plasmodia, and species belonging to the genus Thelohanellus and Henneguya formed intrafilamental vascular (FV) type plasmodia. Mixed infections comprising 2, 3 or 4 different myxozoan species were noted in individual fish. The most common type of parasitism was polyparasitism due to 4 myxobolids co-occuring in fish with an infection rate of 23.16%. All species caused mild to severe haemorrhagic gill disease with little clinical symptomatology.

  10. 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.

  11. From catchment to fish: Impact of anthropogenic pressures on gill histopathology.

    PubMed

    Fonseca, A R; Sanches Fernandes, L F; Fontainhas-Fernandes, A; Monteiro, S M; Pacheco, F A L

    2016-04-15

    Gill histopathology was investigated in barbel (Luciobarbus bocagei) and nase (Pseudochondrostoma sp.) in sub-catchments of Paiva River (Portugal) located upstream and downstream of a Waste Water Treatment Plant (WWTP). Multivariate statistical analyses were performed to set up correlations between the species sample (n=24) and injury types (8). The results discriminate well edema and vasodilatation between reference (upstream) and disturbed (downstream) samples. Using a watershed model, time series of physico-chemical parameters and heavy metal concentrations were calibrated and validated for the entire Paiva River basin as to investigate the relationship between water quality and the gill histopathology results. Increased concentrations of heavy metal downstream, specifically of zinc and lead, coincided with a higher severity of histopathological alterations in the fish gills. Significant but less evident relationship between water quality parameters and severity of gill injuries in the analyzed fish species were also observed for fecal coliforms, water temperature and manganese. Notwithstanding the location of the samples upstream and downstream of the WWTP, contamination of Paiva River and its effect on gill injuries cannot be disconnected from other punctual and diffuse pollution sources acting in different sectors within the watershed, namely agriculture and forest management. The severity of histopathological alterations in the fish gills reflected differences in the type and concentration of contaminants in Paiva River, and consequently can be viewed as valuable indicator of water quality. PMID:26851883

  12. Prevalence, site and tissue preference of myxozoan parasites infecting gills of cultured fish in Punjab (India).

    PubMed

    Kaur, Harpreet; Katoch, Anu

    2016-02-25

    Native carp species cultured in Indian farms in Punjab (catla Catla catla, rohu Labeo rohita, mrigal Cirrhinus mrigala, exotic carps such as silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella, common carp Cyprinus carpio and a catfish Sperata seenghala) were examined for the presence of myxozoan parasites infecting gills. Firstly, the gills were examined under a zoom-stereomicroscope for the presence of plasmodia. The number of plasmodia per gill was counted to determine the index for the intensity of infection. Infected tissues were processed for histology, and 3-4 µm sections of infected gills were stained with haematoxylin & eosin and Luna's method. A total of 19 species of myxosporean were found infecting various cell types in the gills. Of these, 14 species belonged to the genus Myxobolus, 3 species to the genus Thelohanellus and 2 species to the genus Henneguya. Species belonging to the genus Myxobolus formed the interlamellar and intralamellar vascular (LV) type plasmodia, and species belonging to the genus Thelohanellus and Henneguya formed intrafilamental vascular (FV) type plasmodia. Mixed infections comprising 2, 3 or 4 different myxozoan species were noted in individual fish. The most common type of parasitism was polyparasitism due to 4 myxobolids co-occuring in fish with an infection rate of 23.16%. All species caused mild to severe haemorrhagic gill disease with little clinical symptomatology. PMID:26912043

  13. 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.

  14. Gill remodelling during terrestrial acclimation reduces aquatic respiratory function of the amphibious fish Kryptolebias marmoratus.

    PubMed

    Turko, Andy J; Cooper, Chris A; Wright, Patricia A

    2012-11-15

    The skin-breathing amphibious fish Kryptolebias marmoratus experiences rapid environmental changes when moving between water- and air-breathing, but remodelling of respiratory morphology is slower (~1 week). We tested the hypotheses that (1) there is a trade-off in respiratory function of gills displaying aquatic versus terrestrial morphologies and (2) rapidly increased gill ventilation is a mechanism to compensate for reduced aquatic respiratory function. Gill surface area, which varied inversely to the height of the interlamellar cell mass, was increased by acclimating fish for 1 week to air or low ion water, or decreased by acclimating fish for 1 week to hypoxia (~20% dissolved oxygen saturation). Fish were subsequently challenged with acute hypoxia, and gill ventilation or oxygen uptake was measured. Fish with reduced gill surface area increased ventilation at higher dissolved oxygen levels, showed an increased critical partial pressure of oxygen and suffered impaired recovery compared with brackish water control fish. These results indicate that hyperventilation, a rapid compensatory mechanism, was only able to maintain oxygen uptake during moderate hypoxia in fish that had remodelled their gills for land. Thus, fish moving between aquatic and terrestrial habitats may benefit from cutaneously breathing oxygen-rich air, but upon return to water must compensate for a less efficient branchial morphology (mild hypoxia) or suffer impaired respiratory function (severe hypoxia).

  15. From catchment to fish: Impact of anthropogenic pressures on gill histopathology.

    PubMed

    Fonseca, A R; Sanches Fernandes, L F; Fontainhas-Fernandes, A; Monteiro, S M; Pacheco, F A L

    2016-04-15

    Gill histopathology was investigated in barbel (Luciobarbus bocagei) and nase (Pseudochondrostoma sp.) in sub-catchments of Paiva River (Portugal) located upstream and downstream of a Waste Water Treatment Plant (WWTP). Multivariate statistical analyses were performed to set up correlations between the species sample (n=24) and injury types (8). The results discriminate well edema and vasodilatation between reference (upstream) and disturbed (downstream) samples. Using a watershed model, time series of physico-chemical parameters and heavy metal concentrations were calibrated and validated for the entire Paiva River basin as to investigate the relationship between water quality and the gill histopathology results. Increased concentrations of heavy metal downstream, specifically of zinc and lead, coincided with a higher severity of histopathological alterations in the fish gills. Significant but less evident relationship between water quality parameters and severity of gill injuries in the analyzed fish species were also observed for fecal coliforms, water temperature and manganese. Notwithstanding the location of the samples upstream and downstream of the WWTP, contamination of Paiva River and its effect on gill injuries cannot be disconnected from other punctual and diffuse pollution sources acting in different sectors within the watershed, namely agriculture and forest management. The severity of histopathological alterations in the fish gills reflected differences in the type and concentration of contaminants in Paiva River, and consequently can be viewed as valuable indicator of water quality.

  16. Effect of seawater transfer on CYP1A gene expression in rainbow trout gills.

    PubMed

    Leguen, I; Odjo, N; Le Bras, Y; Luthringer, B; Baron, D; Monod, G; Prunet, P

    2010-06-01

    During the transfer of rainbow trout from freshwater to seawater, the gills have to switch from an ion-absorption epithelium to an ion-secretion epithelium in order to maintain equilibrium of their hydromineral balance. After a change to ambient salinity, several gill modifications have already been demonstrated, including ion transporters. In order to identify new branchial mechanisms implicated in seawater acclimation, we carried out an extensive analysis of gene expression in gills using microarray technology. This strategy allowed us to show that CYP1A gene expression was up-regulated in the gills after salinity transfer. This increase was confirmed by real-time reverse transcription PCR. Furthermore, measurements of CYP1A enzyme activity (EROD) showed a significant increase after transfer to seawater. Immunohistochemistry analysis in the gills revealed that cells with a higher expression of CYP1A protein were principally pillar cells and those in the primary lamellae not in contact with the external medium. The results of this study suggest for the first time that CYP1A may be implicated in the seawater acclimation of the gills of rainbow trout.

  17. CD27-CD70 interactions regulate B-cell activation by T cells.

    PubMed Central

    Kobata, T; Jacquot, S; Kozlowski, S; Agematsu, K; Schlossman, S F; Morimoto, C

    1995-01-01

    CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis. PMID:7479974

  18. McPHAC: McGill Planar Hydrogen Atmosphere Code

    NASA Astrophysics Data System (ADS)

    Haakonsen, Christian Bernt; Turner, Monica L.; Tacik, Nick A.; Rutledge, Robert E.

    2012-10-01

    The McGill Planar Hydrogen Atmosphere Code (McPHAC) v1.1 calculates the hydrostatic equilibrium structure and emergent spectrum of an unmagnetized hydrogen atmosphere in the plane-parallel approximation at surface gravities appropriate for neutron stars. McPHAC incorporates several improvements over previous codes for which tabulated model spectra are available: (1) Thomson scattering is treated anisotropically, which is shown to result in a 0.2%-3% correction in the emergent spectral flux across the 0.1-5 keV passband; (2) the McPHAC source code is made available to the community, allowing it to be scrutinized and modified by other researchers wishing to study or extend its capabilities; and (3) the numerical uncertainty resulting from the discrete and iterative solution is studied as a function of photon energy, indicating that McPHAC is capable of producing spectra with numerical uncertainties <0.01%. The accuracy of the spectra may at present be limited to ~1%, but McPHAC enables researchers to study the impact of uncertain inputs and additional physical effects, thereby supporting future efforts to reduce those inaccuracies. Comparison of McPHAC results with spectra from one of the previous model atmosphere codes (NSA) shows agreement to lsim1% near the peaks of the emergent spectra. However, in the Wien tail a significant deficit of flux in the spectra of the previous model is revealed, determined to be due to the previous work not considering large enough optical depths at the highest photon frequencies. The deficit is most significant for spectra with T eff < 105.6 K, though even there it may not be of much practical importance for most observations.

  19. 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.

  20. 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.

  1. 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. PMID:27288994

  2. Complexation of heavy metals by phytochelatins: voltammetric study of the binding of Cd2+ and Zn2+ ions by the phytochelatin (gamma-Glu-Cys)3Gly assisted by multivariate curve resolution.

    PubMed

    Cruz, Boris H; Díaz-Cruz, José Manuel; Ariño, Cristina; Esteban, Miquel

    2005-02-01

    The complexation of Cd2+, Zn2+, and both together with the phytochelatin (gamma-Glu-Cys)3Gly is studied by differential pulse polarography, and data are analyzed by multivariate curve resolution by alternating least squares (MCR-ALS). MCR-ALS yields the respective unitary voltammograms and concentration profiles of the resolved components, which contain information on the relative stabilities and stoichiometries of the formed complexes. The analysis of these results shows, for the Cd2+/(gamma-Glu-Cys)3Gly system, the presence of different kinds of bound Cd2+. For the Zn2+/ (gamma-Glu-Cys)3Gly system, the poor definition of the reduction signals of the complexes prevents a clear discrimination among differently bound Zn2+ ions. Atentative complexation/ electrochemical model is proposed for when both metal ions, Cd2+ and Zn2+, compete toward complexation, and some of the corresponding equilibrium constants are estimated.

  3. 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.

  4. Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress*

    PubMed Central

    Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

    2013-01-01

    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

  5. 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.

  6. Histological alterations in gills of Astyanax aff. bimaculatus caused by acute exposition to zinc.

    PubMed

    dos Santos, Daiane Cristina Marques; da Matta, Sérgio Luis Pinto; de Oliveira, Juraci Alves; dos Santos, Jorge Abdala Dergam

    2012-11-01

    Increasing contamination of aquatic ecosystems by metals has caused various morphological, physiological and biochemical changes in aquatic organisms, and the gills of fish are recognized as indicators of environmental quality. In this context, the present work proposed to study the effects of different concentrations of zinc (Zn) in the histology of gills of yellow tail lambari (Astyanax aff. bimaculatus) after acute exposure. Seventy-two adult males of A. aff. bimaculatus were used, the treatments were six concentrations of Zn: 0; 3; 5; 10; 15; and 20 mg/L of water, by 96 h, and gills, muscle and bone fragments were removed. Fragments of gills were fixed and included, sectioned in a rotary microtome and stained with toluidin blue. Fragments of bone, muscle and gills were dehydrated and digested to quantify the absorption of Zn. The median lethal concentration (LC(50)) 96 h after Zn acute exposure was 10 mg/L of water. Noteworthy, Zn was highly toxic in acute exposure trials starting at the concentration 5 mg/L. The exposure of fish to the metal caused branchial histopathological changes correlated with increasing concentration, caused the death of fish at concentrations of 10, 15 and 20 mg/L. The histological alterations observed in the gills were hyperplasia, lamellar fusion, aneurysm, destruction of the lamellar epithelium, rupture of membrane, deletion of secondary lamellar high, which presented more severity in treatments exposed to the highest concentrations. In conclusion, gills of A. aff. bimaculatus presented profound histological alterations as a result of Zn exposure, and hence, proved to be excellent indicators of environmental contamination.

  7. Alterations in gill structure in tropical reef fishes as a result of elevated temperatures.

    PubMed

    Bowden, A J; Gardiner, N M; Couturier, C S; Stecyk, J A W; Nilsson, G E; Munday, P L; Rummer, J L

    2014-09-01

    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, increase. 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 (representing their seasonal range) 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.

  8. 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

  9. Alterations in gill structure in tropical reef fishes as a result of elevated temperatures.

    PubMed

    Bowden, A J; Gardiner, N M; Couturier, C S; Stecyk, J A W; Nilsson, G E; Munday, P L; Rummer, J L

    2014-09-01

    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, increase. 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 (representing their seasonal range) 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

  10. In vitro effect of various xenobiotics on trout gill cell volume regulation after hypotonic shock.

    PubMed

    Leguen, I; Prunet, P

    2001-08-01

    Their functions and localisation can expose gill cells to volume changes. To maintain their vital functions, these gill cells must regulate their own volume after cellular swelling or shrinkage. Recently, we showed that rainbow trout pavement gill cells in primary culture have the capacity to regulate their own volume after cellular swelling induced by hypotonic shock. This so-called regulatory volume decrease (RVD) is associated with intracellular calcium increase, which occurs as a transient peak followed by a plateau when maintained a hypotonic condition. Return to an isotonic medium restores baseline [Ca2+]i level. In this study, the effect of different xenobiotics on cellular swelling induced RVD and its calcium signal was investigated in trout pavement gill cells in primary culture. These cells were exposed to different pollutants after confluent epithelium was obtained. After 36 h in xenobiotics exposure in vitro, cellular volume and intracellular calcium concentration were measured. Nonylphenol poly- and di-ethoxylate were lethal at concentrations of 10 and 100 microM, respectively. With 10 microM of the diethoxylate form, cells did not die but, unlike non-treated cells, burst during hypotonic shock (2/3rd strength Ringer solution). With 1 microM nonylphenol polyethoxylate (NPnEO), RVD and [Ca2+]i were reduced. Copper (10 and 100 microM) had no significant effect on gill cell volume regulation. However, the heavy metal modified calcium response to hypotonic shock by inhibiting return to baseline level under isotonic conditions. 10 microM prochloraz and 2,4-dichloroaniline had no effect on cell morphology, volume and [Ca2+]i concentration. With 100 microM, however, prochloraz was lethal and dichloroaniline increased baseline [Ca2+]i. These results indicate that the effects observed on gill cells are consistent with the known toxic properties of the molecules tested, thus confirming the validity of primary culture to investigate the toxic effects of

  11. Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

    PubMed Central

    Watson, Rachael; Wearmouth, Emma; McLoughlin, Amy-Claire; Jackson, Arthur; Ward, Sophie; Bertram, Paula; Bennaceur, Karim; Barker, Catriona E.; Pappworth, Isabel Y.; Kavanagh, David; Lea, Susan M.; Atkinson, John P.; Goodship, Timothy H.J.; Marchbank, Kevin J.

    2015-01-01

    Autoantibody formation against Factor H (FH) is found in 7–10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS. PMID:25150608

  12. CD83 and GRASP55 interact in human dendritic cells.

    PubMed

    Stein, Marcello F; Blume, Katja; Heilingloh, Christiane S; Kummer, Mirko; Biesinger, Brigitte; Sticht, Heinrich; Steinkasserer, Alexander

    2015-03-27

    CD83 is one of the best known surface markers for mature human dendritic cells (DCs). The full-length 45 kDa type-I membrane-bound form (mbCD83) is strongly glycosylated upon DCs maturation. As co-stimulatory properties of CD83 are attributed to mbCD83 surface expression is required for efficient T-cell stimulation by mature DCs. By yeast two-hybrid screening, we were able to identify GRASP55 as interaction partner of CD83. DCs maturation induces endogenous CD83 protein expression with simultaneous regulation of CD83 glycosylation, interaction and co-localization with GRASP55 and CD83 surface exposure. GRASP55 is especially known for its role in maintaining Golgi architecture, but also plays a role in Golgi transport of specific cargo proteins bearing a C-terminal valine residue. Here we additionally demonstrate that binding of CD83 and GRASP55 rely on the C-terminal TELV-motif of CD83. Mutation of this TELV-motif not only disrupted binding to GRASP55, but also altered the glycosylation pattern of CD83 and reduced its membrane expression. Here we show for the first time that GRASP55 interacts with CD83 shortly after induction of DC maturation and that this interaction plays a role in CD83 glycosylation as well as in surface expression of CD83 on DCs. PMID:25701785

  13. The Gilles de la Tourette syndrome: the current status.

    PubMed

    Robertson, Mary May

    2012-10-01

    Gilles de la Tourette syndrome (GTS) is characterised by multiple motor and one or more vocal/phonic tics. GTS was once thought to be rare, but many relatively recent studies suggest that the prevalence is about 1% of the worldwide community, apart from in Sub-Saharan Black Africa. Comorbidity and coexistent psychopathology are common, occurring in about 90% of clinical cohorts and individuals in the community. The most common comorbidities are attention deficit hyperactivity disorder, obsessive-compulsive behaviours, and disorder, and autistic spectrum disorders, while the most common coexisting psychopathologies are depression, anxiety and behavioural disorders such as oppositional defiant and conduct disorder. There has been an increasing amount of evidence to show that the quality of life in young people is reduced when compared with normative data or healthy control populations. It is widely accepted that most cases of GTS are inherited, but the genetic mechanisms appear much more complex than previously understood, as evidenced by many recent studies; indeed, there have been suggestions of 'general neurodevelopmental genes' which affect the brain development after which the 'specific GTS gene(s)' may further affect the phenotype. Other aetiopathogenetic suggestions have included environmental factors such as neuro-immunological factors, infections, prenatal and peri-natal difficulties and androgen influences. Few studies have addressed aetiology and phenotype, but initial results are exciting. The search for endophenotypes has followed subsequently. Intriguing neuroanatomical and brain circuitry abnormalities have now been suggested in GTS; the most evidence is for cortical thinning and a reduction in the size of the caudate nucleus. Thorough assessment is imperative and multidisciplinary management is the ideal. Treatment should be 'symptom targeted', and in mild cases, psycho-education and reassurance for the patient and the family may be sufficient

  14. Implications for osmorespiratory compromise by anatomical remodeling in the gills of Arapaima gigas.

    PubMed

    Ramos, Cleverson Agner; Fernandes, Marisa Narciso; da Costa, Oscar Tadeu Ferreira; Duncan, Wallice Paxiuba

    2013-10-01

    The gill structure of the Amazonian fish Arapaima gigas, an obligatory air breather, was investigated during its transition from water breathing to the obligatory air breathing modes of respiration. The gill structure of A. gigas larvae is similar to that of most teleost fish; however, the morphology of the gills changes as the fish grow. The main morphological changes in the gill structure of a growing fish include the following: (1) intense cell proliferation in the filaments and lamellae, resulting in increasing epithelial thickness and decreasing interlamellar distance; (2) pillar cell system atrophy, which reduces the blood circulation through the lamellae; (3) the generation of long cytoplasmic processes from the epithelial cells into the intercellular space, resulting in continuous and sinuous paracellular channels between the epithelial cells of the filament and lamella that may be involved in gas, ion, and nutrient transport to epithelial cells; and (4) intense mitochondria-rich cell (MRC) proliferation in the lamellar epithelium. All of these morphological changes in the gills contribute to a low increase of the respiratory surface area for gas exchange and an increase in the water-blood diffusion distance increasing their dependence on air-breathing as fish developed. The increased proliferation of MRCs may contribute to increased ion uptake, which favors the regulation of ion content and pH equilibrium. PMID:23956000

  15. Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia.

    PubMed

    Valladão, G M R; Pádua, S B; Gallani, S U; Menezes-Filho, R N; Dias-Neto, J; Martins, M L; Ishikawa, M M; Pilarski, F

    2013-11-01

    Trichodinids are ciliated protozoa that are widely known as one of the main groups of fish parasites. The genus Trichodina presents the greatest species diversity. However, records of Paratrichodina species are scarce, and little is known about their pathogenicity in hosts. The present study provides new records of Paratrichodina africana Kazubski and El-Tantawy (1986) in Nile tilapia from South America and descriptions of pathological changes and seasonality. A total of 304 farmed fish were examined. From gill scraping, parasites were identified using Klein's nitrate impregnation method. Gill samples were fixed for histopathological analysis. Small trichodinid found in this study have a prominent blade apophysis and narrow central part and blade shape that corresponds to the characteristics of P. africana Kazubski and El-Tantawy (1986). Gill lesions were proportional to parasite intensity, in which the gill tissue was compromised in heavy infestation. Proliferative disturbances were found, including epithelial hyperplasia, desquamation, and mononuclear and eosinophilic infiltrate that culminated in necrosis. We did not observe a seasonality effect on the occurrence of P. africana. This ciliated protozoan causes compromised respiratory capacity that leads to severe gill lesions and currently is an important pathogen that afflicts intensive tilapia cultures in Brazil.

  16. Clinical and pathological features of common gill diseases of cultured salmonids in Ontario

    PubMed Central

    Speare, David J.; Ferguson, Hugh W.

    1989-01-01

    We reviewed the clinical presentations and histopathology of 118 diagnostic submissions of trout with infectious gill diseases from commercial trout farms within Ontario. Bacterial gill disease (BGD) (56%) and nodular gill disease (NGD) (26.2%) together accounted for 82.2% of these submissions. Submissions of fish with BGD occurred in every month of the year, but were proportionally more frequent in late winter and spring than in late summer and fall. Chemotherapy of groups of fish with gill diseases was common prior to submission, but the success rate of such treatment was low (29% for BGD; 21% for NGD). Specific therapeutic protocols implemented following etiological diagnosis of BGD were successful in 80% of the previously unresponsive cases and in 88.8% of previously untreated cases. The gills of trout collected within 48-96 h of treatment and apparent clinical recovery lacked bacteria and necrotic epithelial cells, but features such as lamellar fusion and epithelial hyperplasia were similar between recovered and affected fish. ImagesFigure 1.Figure 2.Figure 4.Figure 5.Figure 6.Figure 7.Figure 8. PMID:17423456

  17. Gills of hydrothermal vent annelids: Structure, ultrastructure and functional implications in two alvinellid species

    NASA Astrophysics Data System (ADS)

    Jouin, Claude; Gaill, Françoise

    The anatomy, fine structure, diffusion distances and respiratory surface areas of the gills of two species of the polychaete family Alvinellidae have been investigated. Each gill consists of a stem on which are inserted two opposite rows of respiratory elements: flat sickle-shaped lamellae in Alvinella pompejana and cylindrical filaments in Paralvinella grasslei. Both lamellae and filaments have a ciliated mucous epidermis, a central layer of supporting and muscle cells and are devoid of coelomic cavity. Each respiratory element possesses one afferent and one efferent marginal vessel, united to each other distally, and connected proximally to separate longitudinal vessels running in the stem. Superficial parallel blood spaces connect the marginal vessels across the lamella or filament. Deeper, between the basal laminae of the epidermis and that of the central cell layer, a blood sinus is also present. The marginal vessels and the superficial blood spaces actually are intraepidermal extensions of this deep blood sinus. The diffusion distances are very small owing to the intraepidermal position of the respiratory blood spaces. The specific gill surface areas in A. pompejana and P. grasslei are the largest known today in polychaetes, respectively 12 and 47 cm 2 per g wet weight. The distinctive features of the gills are possibly related to a low oxygen content of the ambient seawater. Numerous crystalline granules scattered in the gill epidermis suggest that this epithelium has a detoxifying function.

  18. Gill histopathology of Maria-da-toca Hypleurochilus fissicornis by metacercariae of Bucephalus margaritae (Digenea: Bucephalidae).

    PubMed

    Silva, Renato Z; da Costa Marchiori, Natalia; Magalhães, Aimê Rachel M; Cousin, João Carlos B; Romano, Luis Alberto; Pereira, Joaber

    2016-06-01

    Gills of Maria-da-toca Hypleurochilus fissicornis collected at Ponta do Sambaqui-Florianópolis island-Brazil, were analyzed to describe the histopathology caused by metacercaria of Bucephalus margaritae. Gills were submitted to the routine histological techniques for embedding in paraffin and permanent mounting in Balsam and stereoscopic analysis. Metacercariae showed a branchial infection site pattern for encystations. The branchial infection site pattern is half-basalward in the primary branchial filament with amplitude of the infection of 1-3 metacercaria. Cysts occurred within branchial abductor muscle and cartilaginous and osseous tissues of the gills. Each metacercariae had a contentional hyaline parasitic capsule and melanin-like pigmentation. The half-apicalward region of the primary branchial filaments showed several dysplasia degrees, cartilage and osseous degeneration (pyknosis), thrombosis and immune exudated cells (mainly lymphocytes). Cytopathologies as thickening of the epithelium lining of the secondary branchial filaments were a response of the branchial infection site pattern of the metacercaria. Interlamellar obliteration and fusion of the lamellae due to the hypertrophy and hyperplasia of the epithelial lining as well as chloride cells occurred. Pyknosis of pillar cells and epithelial lining cells from the secondary branchial filaments were also present. Bucephalosis in H. fissicornis gills is no-hemorrhagic and no-fatal branchitis, but could compromises the gill functions and could permits the secondary opportunistic infections.

  19. 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.

  20. Gill histopathology of Maria-da-toca Hypleurochilus fissicornis by metacercariae of Bucephalus margaritae (Digenea: Bucephalidae).

    PubMed

    Silva, Renato Z; da Costa Marchiori, Natalia; Magalhães, Aimê Rachel M; Cousin, João Carlos B; Romano, Luis Alberto; Pereira, Joaber

    2016-06-01

    Gills of Maria-da-toca Hypleurochilus fissicornis collected at Ponta do Sambaqui-Florianópolis island-Brazil, were analyzed to describe the histopathology caused by metacercaria of Bucephalus margaritae. Gills were submitted to the routine histological techniques for embedding in paraffin and permanent mounting in Balsam and stereoscopic analysis. Metacercariae showed a branchial infection site pattern for encystations. The branchial infection site pattern is half-basalward in the primary branchial filament with amplitude of the infection of 1-3 metacercaria. Cysts occurred within branchial abductor muscle and cartilaginous and osseous tissues of the gills. Each metacercariae had a contentional hyaline parasitic capsule and melanin-like pigmentation. The half-apicalward region of the primary branchial filaments showed several dysplasia degrees, cartilage and osseous degeneration (pyknosis), thrombosis and immune exudated cells (mainly lymphocytes). Cytopathologies as thickening of the epithelium lining of the secondary branchial filaments were a response of the branchial infection site pattern of the metacercaria. Interlamellar obliteration and fusion of the lamellae due to the hypertrophy and hyperplasia of the epithelial lining as well as chloride cells occurred. Pyknosis of pillar cells and epithelial lining cells from the secondary branchial filaments were also present. Bucephalosis in H. fissicornis gills is no-hemorrhagic and no-fatal branchitis, but could compromises the gill functions and could permits the secondary opportunistic infections. PMID:27413297

  1. 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.

  2. 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.

  3. Prolactin regulates transcription of the ion uptake Na+/Cl− cotransporter (ncc) gene in zebrafish gill

    PubMed Central

    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. PMID:23395804

  4. 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

  5. Effect of hypotonic shock on cultured pavement cells from freshwater or seawater rainbow trout gills.

    PubMed

    Leguen, Isabelle; Prunet, Patrick

    2004-02-01

    The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca(2+) with an initial peak followed by a sustained plateau. Absence of external Ca(2+) did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca(2+)](i) response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca(2+) signalling response to hypotonic shock was observed regardless of the presence or absence of Ca(2+) in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.

  6. Mercury-binding proteins of Mytilus edulis

    SciTech Connect

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  7. Cytokine Responses in Gills of Capoeta umbla as Biomarkers of Environmental Pollution.

    PubMed

    Danabas, Durali; Yildirim, Nuran Cikcikoglu; Yildirim, Numan; Onal, Ayten Oztufekci; Uslu, Gulsad; Unlu, Erhan; Danabas, Seval; Ergin, Cemil; Tayhan, Nilgun

    2016-03-01

    Immunological biomarkers reflect the effects of exposure to environmental contaminants. In this study, the suitability and sensitivity of cytokine responses, interleukin1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) in gill tissues of Capoeta umbla (Heckel, 1843), collected from different regions, as early warning indices of environmental pollution and ecosystem health was evaluated. Fish and water samples were taken from ten stations in March and September 2011 and 2012. Tumor necrosis factor-α, IL-1β and IL-6 levels were determined in samples of the gill tissues by using an ELISA kit. Significant variations of TNF-α, IL-1β and IL-6 levels observed between stations and seasons. The results of this study show that seasonal variations of cytokine responses in gills of Capoeta umbla are sensitive to the contaminants present in Uzuncayir Dam Lake (Tunceli, Turkey) water and are valuable biomarkers for environmental pollution and ecosystem health.

  8. Histopathological effects of atrazine on gills of Caspian kutum Rutilus frisii kutum fingerlings.

    PubMed

    Khoshnood, Zahra; Jamili, Shahla; Khodabandeh, Saber

    2015-04-01

    The use of chemical pesticides has increased environmental pollution and affects fishes as non-target organisms. To investigate the toxic effects of the widely used herbicide atrazine on Caspian kutum Rutilus frisii kutum fingerlings, fish were exposed to a sublethal concentration of half LC50 for 96 h. The main alterations visible in the gill tissue were detachment of the epithelium of the lamellae, necrosis, lamellar fusion, hyperplasia, club shaped lamellae, collapse of the lamellae, shrinkage and curling of the lamellae, and ultrastructural alterations such as necrosis of the apical microridges of the pavement cells. Results also showed that the gill ionocytes were fewer in number and larger in size in the atrazine-exposed fish. Atrazine appears to be highly toxic to Caspian kutum fingerlings even at a sublethal concentration (12.47 mg l(-1)) and acute exposure. This toxicity could affect gill respiration and ion regulation function of fingerlings by damaging tissue, pavement cells, and ionocytes. PMID:25850400

  9. Cytokine Responses in Gills of Capoeta umbla as Biomarkers of Environmental Pollution.

    PubMed

    Danabas, Durali; Yildirim, Nuran Cikcikoglu; Yildirim, Numan; Onal, Ayten Oztufekci; Uslu, Gulsad; Unlu, Erhan; Danabas, Seval; Ergin, Cemil; Tayhan, Nilgun

    2016-03-01

    Immunological biomarkers reflect the effects of exposure to environmental contaminants. In this study, the suitability and sensitivity of cytokine responses, interleukin1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) in gill tissues of Capoeta umbla (Heckel, 1843), collected from different regions, as early warning indices of environmental pollution and ecosystem health was evaluated. Fish and water samples were taken from ten stations in March and September 2011 and 2012. Tumor necrosis factor-α, IL-1β and IL-6 levels were determined in samples of the gill tissues by using an ELISA kit. Significant variations of TNF-α, IL-1β and IL-6 levels observed between stations and seasons. The results of this study show that seasonal variations of cytokine responses in gills of Capoeta umbla are sensitive to the contaminants present in Uzuncayir Dam Lake (Tunceli, Turkey) water and are valuable biomarkers for environmental pollution and ecosystem health. PMID:26931532

  10. The K-Gill - A twin propeller-vane anemometer for measurements of atmospheric turbulence

    NASA Technical Reports Server (NTRS)

    Atakturk, Serhad S.; Katsaros, Kristina B.

    1989-01-01

    A twin propeller-vane anemometer developed at the University of British Columbia was successfully used for unattended measurement of atmospheric turbulence over extended periods of time during moderate to high winds in the presence of sea spray. In this paper, a new design in twin propeller-vane anemometers is introduced. The instrument consists of two Gill anemometers mounted on a vane. The propeller shafts, one pointing up and the other pointing down, make an angle of 45 deg with the horizontal (hence, the name K-Gill). In addition to the desirable characteristics of the earlier design the K-Gill has symmetry, so that updraft and downdraft winds are measured with equal sensitivity. An algorithm for obtaining the vertical and downstream horizontal components of the wind velocity is described. Various sources of error and their magnitudes are discussed.

  11. 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