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Sample records for adhesin psrp binds

  1. PSRP Approach to PRA

    Science.gov (United States)

    Stewart, Christine

    2004-01-01

    There have been two scenarios in which the Payload Safety Review Panel (PSRP) has considered a Probabilistic Risk Assessment (PRA) a viable tool. For use in considering trade-offs during the initial design. When in a waiver condition to explain rationale for acceptance. The PSRP would consider the use of PRA in other applications where credible.

  2. Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

    Science.gov (United States)

    Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-04-01

    Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

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    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  4. Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity.

    Science.gov (United States)

    Fardini, Yann; Wang, Xiaowei; Témoin, Stéphanie; Nithianantham, Stanley; Lee, David; Shoham, Menachem; Han, Yiping W

    2011-12-01

    Fusobacterium nucleatum is a Gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA colocalized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and Escherichia coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an 'enabler' for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens. © 2011 Blackwell Publishing Ltd.

  5. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

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    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M. (VA); (UCLA); (Vanderbilt); (UCSF)

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  6. Heterologous expression of the Treponema pallidum laminin-binding adhesin Tp0751 in the culturable spirochete Treponema phagedenis.

    Science.gov (United States)

    Cameron, Caroline E; Kuroiwa, Janelle M Y; Yamada, Mitsunori; Francescutti, Teresa; Chi, Bo; Kuramitsu, Howard K

    2008-04-01

    Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.

  7. Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

    Science.gov (United States)

    Hanski, E; Caparon, M

    1992-07-01

    Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

  8. The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori.

    Science.gov (United States)

    Pang, Siew Siew; Nguyen, Stanley Thai Son; Perry, Andrew J; Day, Christopher J; Panjikar, Santosh; Tiralongo, Joe; Whisstock, James C; Kwok, Terry

    2014-03-07

    The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

  9. Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

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    Previato Jose O

    2008-05-01

    Full Text Available Abstract Background The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs. In the present study, nuclear magnetic resonance (NMR was used to map the binding site(s of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. Results The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. Conclusion Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

  10. Bioaccumulation of heavy metals by fimbrial designer adhesins

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, Kristian; Klemm, Per

    1999-01-01

    Naturally occurring adhesins bind to specific molecular targets in a lock-and-key fashion due to the composition of the binding domain of the adhesin. By introduction of random peptide libraries in a suitable surface exposed carrier protein it is possible to create and select designer adhesins wi...

  11. Molecular simulations of lactose-bound and unbound forms of the FaeG adhesin reveal critical amino acids involved in sugar binding.

    Science.gov (United States)

    Baker, Joseph L; Jafri, Heba

    2016-11-01

    F4 fimbriae are protein filaments found in enterotoxigenic Escherichia coli cells and are implicated in the process of bacterial infection due to their function as bacterial adhesins. These filaments are comprised from several proteins, but the bacterial adhesin FaeG, which is a lactose-binding protein, is the major subunit comprising F4 fimbriae. Crystal structures for three variants of the FaeG protein were recently solved, including the ad variant of FaeG that was crystallized in complex with lactose. However, the dynamics of the FaeG protein bound to lactose have not been explored previously using molecular dynamics simulations. Therefore, in order to study the dynamical interactions between the FaeG ad variant and lactose, we have carried out the first all-atom molecular dynamics simulations of this system. We have also probed the role of crystallographic water molecules on the stability of lactose in the FaeG binding site, and have simulated seven FaeG mutants to probe the influence of amino acid substitutions on the ability of FaeG to bind lactose effectively. Our simulations agree well with experimental results for the influence of mutations on lactose binding, provide dynamical insights into the interactions of FaeG with lactose, and also suggest the possibility of additional regions of the FaeG protein that may act as secondary lactose binding sites. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. EHEC Adhesins

    Science.gov (United States)

    McWilliams, Brian D.; Torres, Alfredo G.

    2014-01-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbriae) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this chapter have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:25635238

  13. Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

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    Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2015-03-01

    Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. © 2014 John Wiley & Sons Ltd.

  14. Vitronectin Binds to a Specific Stretch within the Head Region of Yersinia Adhesin A and Thereby Modulates Yersinia enterocolitica Host Interaction.

    Science.gov (United States)

    Mühlenkamp, Melanie C; Hallström, Teresia; Autenrieth, Ingo B; Bohn, Erwin; Linke, Dirk; Rinker, Janina; Riesbeck, Kristian; Singh, Birendra; Leo, Jack C; Hammerschmidt, Sven; Zipfel, Peter F; Schütz, Monika S

    2017-01-01

    Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis. © 2016 S. Karger AG, Basel.

  15. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

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    Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

    2016-02-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  16. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N.; Crowley, Paula J.; Brady, L. Jeannine; Long, Joanna R.

    2016-01-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin

  17. More than a marine propeller--the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus.

    Science.gov (United States)

    Troge, Anja; Scheppach, Wolfgang; Schroeder, Bjoern O; Rund, Stefan A; Heuner, Klaus; Wehkamp, Jan; Stange, Eduard F; Oelschlaeger, Tobias A

    2012-12-01

    The flagellum of the probiotic Escherichia coli strain Nissle 1917 (EcN) is not just responsible for motility, but also for EcN's ability to induce the production of human β-defensin 2. Here, we report a third function of this EcN organell. In this study we investigated the role of the EcN flagellum in adhesion to different host tissues by ex vivo and in vitro studies. Ex vivo studies with cryosections of human gut biopsies revealed that the flagellum of EcN is most likely important for efficient adhesion to the human intestinal tract. These results and in vitro studies with different epithelial cells indicated that the presence of mucus is important for efficient mediation of adhesion by the flagellum of EcN. We observed direct interaction between isolated flagella from EcN wild type and porcine mucin 2 as well as human mucus. However, we could not observe any interaction of the flagella with murine mucus. For the first time, we identified the mucus component gluconate as one receptor for the binding of flagella from EcN and were able to exclude the flagellin domain D3 as a responsible interaction partner. We propose that the flagellum of EcN is its major adhesin in vivo, which enables this probiotic strain to compete efficiently for binding sites on host tissue with several bacterial pathogens. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

    2016-02-15

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  19. Strain-specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete.

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    Yi-Pin Lin

    2014-07-01

    Full Text Available Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.

  20. Strain-specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete.

    Science.gov (United States)

    Lin, Yi-Pin; Benoit, Vivian; Yang, Xiuli; Martínez-Herranz, Raúl; Pal, Utpal; Leong, John M

    2014-07-01

    Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.

  1. Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence.

    Directory of Open Access Journals (Sweden)

    Carlos J Sanchez

    Full Text Available We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4 mutants expressing the capsule types 6A (+6A, 7F (+7F and 23F (+23F as well as a deleted and restored serotype 4 (+4 control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA, Choline binding protein A (CbpA, and Pneumococcal serine-rich repeat protein (PsrP changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.

  2. Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

    Energy Technology Data Exchange (ETDEWEB)

    Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.; Cecchini, Gary; Sullam, Paul M.; Iverson, T.M. (VA); (Vanderbilt); (UCSF)

    2012-07-11

    The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB{sub BR}) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB{sub BR} buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB{sub BR} in each buffer. While both sets of conditions supported crystal growth in space group P2{sub 1}2{sub 1}2{sub 1}, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 {angstrom} for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 {angstrom} for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 {angstrom} resolution. A complete data set has been collected to 1.3 {angstrom} resolution with an overall R{sub merge} value of 0.04 and an R{sub merge} value of 0.33 in the highest resolution shell.

  3. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  4. Tribological performance of paddy straw reinforced polypropylene (PSRP and unidirectional glass-pultruded-kenaf (UGPK composites

    Directory of Open Access Journals (Sweden)

    R.M. Nasir

    2014-06-01

    Full Text Available In standard preparation and fabrication of natural-fibre embedded composites, 5 wt. % of natural fibre is enough to strengthen and homogenized in parental matrix as the mechanical strength was observed to increase by more than 25% of pure matrix. Hence, paddy straw and kenaf has been a potential candidate in northern region of Malaysia due to its abundance and easily replenished. A unidirectional glass-pultruded-kenaf (UGPK and paddy straws reinforced polypropylene (PSRP was studied focusing on its tribological performance. Meanwhile, friction and wear properties were examined using pin-on-disc machine under ambient temperature with dry contact condition. The tests were conducted at various sliding velocities (1.178-2.749m/s and applied normal loads (9.82-19.64N. The results showed that specific wear rate and friction coefficient decreased with increasing applied normal load and sliding velocity, but the applied normal load was more influential. The coefficient of friction ranges from 0.5 to 4 and wear rate varies from 0.5 to 4 x 10-5 mm3/Nm for PSRP. The friction coefficient of UGPK is within a range of 2.76 to 4.54 at the given test parameters while its wear rate ranging from 0.8 to 1.79 x 10-5mm3/Nm. The failure mode observed during the test was micro-buckling and followed by splitting while fiber-matrix interfacial failure occurred.

  5. Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

    Directory of Open Access Journals (Sweden)

    Sanjiv Kumar

    Full Text Available Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA, Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase and Rv0309 (L,D-transpeptidase bind to fibronectin and laminin. We report Rv2599 (membrane protein, Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.

  6. The Biology of Neisseria Adhesins

    Directory of Open Access Journals (Sweden)

    Miao-Chiu Hung

    2013-07-01

    Full Text Available Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology.

  7. The Giant Adhesin SiiE of Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Britta Barlag

    2015-01-01

    Full Text Available Salmonella enterica is a Gram-negative, food-borne pathogen, which colonizes the intestinal tract and invades enterocytes. Invasion of polarized cells depends on the SPI1-encoded type III secretion system (T3SS and the SPI4-encoded type I secretion system (T1SS. The substrate of this T1SS is the non-fimbrial giant adhesin SiiE. With a size of 595 kDa, SiiE is the largest protein of the Salmonella proteome and consists of 53 repetitive bacterial immunoglobulin (BIg domains, each containing several conserved residues. As known for other T1SS substrates, such as E. coli HlyA, Ca2+ ions bound by conserved D residues within the BIg domains stabilize the protein and facilitate secretion. The adhesin SiiE mediates the first contact to the host cell and thereby positions the SPI1-T3SS to initiate the translocation of a cocktail of effector proteins. This leads to actin remodeling, membrane ruffle formation and bacterial internalization. SiiE binds to host cell apical membranes in a lectin-like manner. GlcNAc and α2–3 linked sialic acid-containing structures are ligands of SiiE. Since SiiE shows repetitive domain architecture, we propose a zipper-like binding mediated by each individual BIg domain. In this review, we discuss the characteristics of the SPI4-T1SS and the giant adhesin SiiE.

  8. Three Yersinia pestis adhesins facilitate Yop delivery to eukaryotic cells and contribute to plague virulence.

    Science.gov (United States)

    Felek, Suleyman; Tsang, Tiffany M; Krukonis, Eric S

    2010-10-01

    To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.

  9. Host determinants of expression of the helicobacter pylori BabA adhesin

    Science.gov (United States)

    Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. BabA is highly polymorphic genetically and functionally among different clinical is...

  10. Valency conversion in the type 1 fimbrial adhesin of Escherichia coli

    DEFF Research Database (Denmark)

    Sokurenko, E.V.; Schembri, Mark; Trintchina, E.

    2001-01-01

    FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate...... that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability...... phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting...

  11. Oil fuel delivery optimization for multi product and multi depot: the case of petrol station replenishment problem (PSRP)

    Science.gov (United States)

    Surjandari, Isti; Rachman, Amar; Dianawati, Fauzia; Wibowo, R. Pramono

    2011-10-01

    With the Oil and Gas Law No. 22 of 2001, national and foreign private enterprises can invest in all sectors of Oil and Gas in Indonesia. In anticipation of this free competition, Pertamina, as a state-owned enterprises, which previously had monopolized the oil and gas business activities in Indonesia, should be able to improve services as well as the efficiency in order to compete in the free market, especially in terms of cost efficiency of fuel distribution to gas station (SPBU). To optimize the distribution activity, it is necessary to design a scheduling system and its fuel delivery routes daily to every SPBU. The determination of routes and scheduling delivery of fuel to the SPBU can be modeled as a Petrol Station Replenishment Problem (PSRP) with the multi-depot, multi-product, time windows and split deliveries, which in this study will be completed by the Tabu Search algorithm (TS). This study was conducted in the area of Bandung, the capital of West Java province, which is a big city and the neighboring city of Jakarta, the capital city of Indonesia. By using the fuel delivery data for one day, the results showed a decrease of 16.38% of the distance of the route compared to the current conditions, which impacted on the reduction of distribution costs and decrease the number of total trips by 5.22% and 3.83%.

  12. Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis.

    Science.gov (United States)

    Tuomanen, E

    1986-12-01

    Two proteins secreted by Bordetella pertussis are known to mediate adherence of these bacteria to mammalian respiratory cilia. When either ciliated cells or other pathogenic bacteria were pretreated with these adhesins, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus acquired the ability to adhere to cilia in vitro and in vivo. Such piracy of adhesins may contribute to superinfection in mucosal diseases such as whooping cough.

  13. Regions important for the adhesin activity of Moraxella catarrhalis Hag

    Directory of Open Access Journals (Sweden)

    Lafontaine Eric R

    2007-07-01

    Full Text Available Abstract Background The Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells. Results The present study demonstrates that adherence of M. catarrhalis isogenic hag mutant strains to the human epithelial cell lines Chang (conjunctival and NCIH292 (lung is reduced by 50–93%. Furthermore, expressing Hag in a heterologous Escherichia coli background substantially increased the adherence of recombinant bacteria to NCIH292 cells and murine type IV collagen. Hag did not, however, increase the attachment of E. coli to Chang cells. These results indicate that Hag directly mediates adherence to NCIH292 lung cells and collagen, but is not sufficient to confer binding to conjunctival monolayers. Several in-frame deletions were engineered within the hag gene of M. catarrhalis strain O35E and the resulting proteins were tested for their ability to mediate binding to NCIH292 monolayers, middle ear cells, and type IV collagen. These experiments revealed that epithelial cell and collagen binding properties are separable, and that residues 385–705 of this ~2,000 amino acid protein are important for adherence to middle ear and NCIH292 cells. The region of O35E-Hag encompassing aa 706 to 1194 was also found to be required for adherence to collagen. In contrast, β-roll repeats present in Hag, which are structural features conserved in several Oca adhesins and responsible for the adhesive properties of Yersinia enterocolitica YadA, are not important for Hag-mediated adherence. Conclusion Hag is a major adherence factor for human cells derived from various anatomical sites relevant to pathogenesis by M. catarrhalis and its structure-function relationships differ from those of other, closely-related autotransporter proteins.

  14. Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii.

    Science.gov (United States)

    Chen, Yu; Bensing, Barbara A; Seepersaud, Ravin; Mi, Wei; Liao, Maofu; Jeffrey, Philip D; Shajahan, Asif; Sonon, Roberto N; Azadi, Parastoo; Sullam, Paul M; Rapoport, Tom A

    2018-04-06

    Many pathogenic bacteria, including Streptococcus gordonii , possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O -glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O -glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N -acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

  15. Characterization of Fusobacterium nucleatum ATCC 23726 adhesins involved in strain-specific attachment to Porphyromonas gingivalis

    Science.gov (United States)

    Park, Jane; Shokeen, Bhumika; Haake, Susan K; Lux, Renate

    2016-01-01

    Bacterial adherence is an essential virulence factor in pathogenesis and infection. Fusobacterium nucleatum has a central role in oral biofilm architecture by acting as a bridge between early Gram-positive and late Gram-negative colonizers that do not otherwise adhere to each other. In this study, we survey a key adherence interaction of F. nucleatum with Porphyromonas gingivalis, and present evidence that multiple fusobacterial adhesins have a role in the attachment of F. nucleatum ATCC 23726 to P. gingivalis in a highly strain-dependent manner. Interaction between these species displayed varying sensitivities to arginine, galactose and lactose. Arginine was found to hamper coaggregation by at least 62% and up to 89% with several P. gingivalis strains and galactose inhibition ranged from no inhibition up to 58% with the same P. gingivalis strains. Lactose consistently inhibited F. nucleatum interaction with these P. gingivalis strains ranging from 40% to 56% decrease in coaggregation. Among the adhesins involved are the previously described Fap2 and surprisingly, RadD, which was described in an earlier study for its function in attachment of F. nucleatum to Gram-positive species. We also provide evidence for the presence of at least one additional adhesin that is sensitive to arginine but unlike Fap2 and RadD, is not a member of the autotransporter family type of fusobacterial large outer membrane proteins. The strain-specific binding profile of multiple fusobacterial adhesins to P. gingivalis highlights the heterogeneity and complexity of interspecies interactions in the oral cavity.

  16. Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli:

    DEFF Research Database (Denmark)

    Sherlock, Orla; Schembri, Mark; Reisner, A.

    2004-01-01

    Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA...... is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form...... binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact...

  17. Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG ▿

    Science.gov (United States)

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos; de Vos, Willem M.; Palva, Airi

    2011-01-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic. PMID:21602388

  18. Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG.

    Science.gov (United States)

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C J; Vanderleyden, Jos; de Vos, Willem M; Palva, Airi

    2011-07-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.

  19. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

    Directory of Open Access Journals (Sweden)

    Mariana Noelia Viale

    2014-01-01

    Full Text Available The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP by host cells are fibronectin (FN dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

  20. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    Science.gov (United States)

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  1. Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp

    Science.gov (United States)

    Engstrom, Michael D.

    2016-01-01

    Urinary tract infections (UTIs) are a major burden to human health. The overwhelming majority of UTIs are caused by uropathogenic Escherichia coli (UPEC) strains. Unlike some pathogens, UPEC strains do not have a fixed core set of virulence and fitness factors but do have a variety of adhesins and regulatory pathways. One such UPEC adhesin is the nonfimbrial adhesin TosA, which mediates adherence to the epithelium of the upper urinary tract. The tos operon is AT rich, resides on pathogenicity island aspV, and is not expressed under laboratory conditions. Because of this, we hypothesized that tosA expression is silenced by H-NS. Lrp, based on its prominent function in the regulation of other adhesins, is also hypothesized to contribute to tos operon regulation. Using a variety of in vitro techniques, we mapped both the tos operon promoter and TosR binding sites. We have now identified TosR as a dual regulator of the tos operon, which could control the tos operon in association with H-NS and Lrp. H-NS is a negative regulator of the tos operon, and Lrp positively regulates the tos operon. Exogenous leucine also inhibits Lrp-mediated tos operon positive regulation. In addition, TosR binds to the pap operon, which encodes another important UPEC adhesin, P fimbria. Induction of TosR synthesis reduces production of P fimbria. These studies advance our knowledge of regulation of adhesin expression associated with uropathogen colonization of a host. PMID:26755158

  2. Capsule shields the function of short bacterial adhesins

    DEFF Research Database (Denmark)

    Schembri, Mark; Dalsgaard, D.; Klemm, Per

    2004-01-01

    occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins......Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to Escherichia coli but can....... This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms....

  3. Capsule Shields the Function of Short Bacterial Adhesins

    OpenAIRE

    Schembri, Mark A.; Dalsgaard, Dorte; Klemm, Per

    2004-01-01

    Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that cap...

  4. Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp.

    Science.gov (United States)

    Engstrom, Michael D; Mobley, Harry L T

    2016-01-11

    Urinary tract infections (UTIs) are a major burden to human health. The overwhelming majority of UTIs are caused by uropathogenic Escherichia coli (UPEC) strains. Unlike some pathogens, UPEC strains do not have a fixed core set of virulence and fitness factors but do have a variety of adhesins and regulatory pathways. One such UPEC adhesin is the nonfimbrial adhesin TosA, which mediates adherence to the epithelium of the upper urinary tract. The tos operon is AT rich, resides on pathogenicity island aspV, and is not expressed under laboratory conditions. Because of this, we hypothesized that tosA expression is silenced by H-NS. Lrp, based on its prominent function in the regulation of other adhesins, is also hypothesized to contribute to tos operon regulation. Using a variety of in vitro techniques, we mapped both the tos operon promoter and TosR binding sites. We have now identified TosR as a dual regulator of the tos operon, which could control the tos operon in association with H-NS and Lrp. H-NS is a negative regulator of the tos operon, and Lrp positively regulates the tos operon. Exogenous leucine also inhibits Lrp-mediated tos operon positive regulation. In addition, TosR binds to the pap operon, which encodes another important UPEC adhesin, P fimbria. Induction of TosR synthesis reduces production of P fimbria. These studies advance our knowledge of regulation of adhesin expression associated with uropathogen colonization of a host. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Fap2 of Fusobacterium nucleatum is a galactose-inhibitable adhesin involved in coaggregation, cell adhesion, and preterm birth.

    Science.gov (United States)

    Coppenhagen-Glazer, S; Sol, A; Abed, J; Naor, R; Zhang, X; Han, Y W; Bachrach, G

    2015-03-01

    Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

    Science.gov (United States)

    Fusco, William G; Choudhary, Neelima R; Routh, Patty A; Ventevogel, Melissa S; Smith, Valerie A; Koch, Gary G; Almond, Glen W; Orndorff, Paul E; Sempowski, Gregory D; Leduc, Isabelle

    2014-06-24

    Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

    Science.gov (United States)

    Kisiela, Dagmara I.; Chattopadhyay, Sujay; Libby, Stephen J.; Karlinsey, Joyce E.; Fang, Ferric C.; Tchesnokova, Veronika; Kramer, Jeremy J.; Beskhlebnaya, Viktoriya; Samadpour, Mansour; Grzymajlo, Krzysztof; Ugorski, Maciej; Lankau, Emily W.; Mackie, Roderick I.; Clegg, Steven; Sokurenko, Evgeni V.

    2012-01-01

    Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella. PMID:22685400

  8. Yersinia infection tools – characterization of structure and function of adhesins

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    Kornelia Malgorzata Mikula

    2013-01-01

    Full Text Available Among the seventeen species of the Gram-negative genus Yersinia, three have been shown to be virulent and pathogenic to humans and animals - Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. In order to be so, they are armoured with various factors that help them adhere to tissues and organelles, cross the cellular barrier and escape the immune system during host invasion. The group of proteins that mediate pathogen-host interactions constitute adhesins. Invasin, Ail, YadA, YadB, YadC, Pla and pH 6 antigen belong to the most prominent and best-known Yersinia adhesins. They act at different times and stages of infection complementing each other by their ability to bind a variety of host molecules such as collagen, fibronectin, laminin, β1 integrins, and complement regulators. All the proteins are anchored in the bacterial outer membrane, often forming rod-like or fimbrial-like structures that protrude to the extracellular milieu. Structural studies have shown that the anchor region forms a β-barrel composed of 8, 10 or 12 antiparallel β strands. Depending on the protein, the extracellular part can be composed of several domains belonging to the immunoglobulin fold superfamily, or form a coiled-coil structure with globular head domain at the end, or just constitute several loops connecting individual β stands in the β-barrel. Those extracellular regions define the activity of each adhesin. This review focuses on the structure and function of these important molecules, and their role in pathogenesis.

  9. Yersinia infection tools—characterization of structure and function of adhesins

    Science.gov (United States)

    Mikula, Kornelia M.; Kolodziejczyk, Robert; Goldman, Adrian

    2013-01-01

    Among the seventeen species of the Gram-negative genus Yersinia, three have been shown to be virulent and pathogenic to humans and animals—Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. In order to be so, they are armoured with various factors that help them adhere to tissues and organelles, cross the cellular barrier and escape the immune system during host invasion. The group of proteins that mediate pathogen–host interactions constitute adhesins. Invasin, Ail, YadA, YadB, YadC, Pla, and pH 6 antigen belong to the most prominent and best-known Yersinia adhesins. They act at different times and stages of infection complementing each other by their ability to bind a variety of host molecules such as collagen, fibronectin, laminin, β1 integrins, and complement regulators. All the proteins are anchored in the bacterial outer membrane (OM), often forming rod-like or fimbrial-like structures that protrude to the extracellular milieu. Structural studies have shown that the anchor region forms a β-barrel composed of 8, 10, or 12 antiparallel β-strands. Depending on the protein, the extracellular part can be composed of several domains belonging to the immunoglobulin fold superfamily, or form a coiled-coil structure with globular head domain at the end, or just constitute several loops connecting individual β-strands in the β-barrel. Those extracellular regions define the activity of each adhesin. This review focuses on the structure and function of these important molecules, and their role in pathogenesis. PMID:23316485

  10. Surface adhesins and exopolymers of selected foodborne pathogens

    DEFF Research Database (Denmark)

    Jaglic, Zoran; Desvaux, Mickaël; Weiss, Agnes

    2014-01-01

    of bacterial surface structures are involved in this process and these promote bacterial adhesion in a more or less specific manner. In this review, we will focus on those surface adhesins and exopolymers in selected foodborne pathogens that are involved mainly in primary adhesion. Their role in biofilm...

  11. Determination of the mimic epitope of the M-like protein adhesin in swine Streptococcus equi subsp. zooepidemicus

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    Tang Fuyu

    2008-10-01

    Full Text Available Abstract Background The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus. Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells. Methods The gene encoding SzP was expressed in E. coli, and the purified recombinant SzP (rSzP was recognized by rabbit anti-S. zooepidemicus antibodies using immunoblot. Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8. Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide. Results Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones. Conclusion Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of S. zooepidemicus strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.

  12. Lactobacillus reuteri Surface Mucus Adhesins Upregulate Inflammatory Responses Through Interactions With Innate C-Type Lectin Receptors.

    Science.gov (United States)

    Bene, Krisztián P; Kavanaugh, Devon W; Leclaire, Charlotte; Gunning, Allan P; MacKenzie, Donald A; Wittmann, Alexandra; Young, Ian D; Kawasaki, Norihito; Rajnavolgyi, Eva; Juge, Nathalie

    2017-01-01

    The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB, upon interaction of L. reuteri ATCC PTA 6475 and ATCC 53608 strains with human monocyte-derived dendritic cells (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also indicated that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly, in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by atomic force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins.

  13. Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

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    A. Patrick Gunning

    2016-11-01

    Full Text Available The mucus layer covering the gastrointestinal (GI epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB, a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3, a mammalian lectin characterised by a single carbohydrate binding domain (CRD. The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

  14. Detection specificity studies of bacteriophage adhesin-coated long-period grating-based biosensor

    Science.gov (United States)

    Koba, Marcin; Śmietana, Mateusz; Brzozowska, Ewa; Górska, Sabina; Mikulic, Predrag; Cusano, Andrea; Bock, Wojtek J.

    2015-09-01

    In this work, we present a label-free detection specificity study of an optical fiber long-period grating (LPG) biosensor working near the dispersion turning point of higher order cladding modes. The LPG sensor functionalized with bacteriophage adhesin is tested with specific and non-specific bacteria dry weight. We show that such biosensor is able to selectively bind, thus recognize different bacteria. We use bacteria dry weights of E. coli B as positive test and E. coli K12 and Salmonella enterica as negative tests. The resonance wavelength shift induced by E. coli B reaches over 90 nm, while for E. coli K12 and Salmonella enterica approximately 40 and 20 nm, respectively.

  15. The role of Actinobacillus actinomycetemcomitans fimbrial adhesin on MMP-8 activity in aggressive periodontitis pathogenesis

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    Rini Devijanti Ridwan

    2012-12-01

    Full Text Available Background: Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans is Gram negative and a major bacterial agent associated with aggressive periodontitis in young adult, this bacteria was an important factor in pathogenesis of aggressive periodontitis. A. actinomycetemcomitans possesses fimbriae with an adhesin protein that was the first bacterial molecules to make physical contact with host. Purpose: The objective of this research was to analyzed the influence of A. actinomycetemcomitans fimbrial adhesin protein induction on MMP-8 activity. Methods: The research was an experimental laboratory study, the step in this study were isolation and identification A. actinomycetemcomitans, characterize A. actinomycetemcomitans adhesin and study the role of A. actinomycetemcomitans adhesin in Wistar rats. Results: The result of this research on the role of adhesin in Wistar rats after analysis with Analysis of Variance (ANOVA showed significant differences in the control group with group induction with A. actinomycetemcomitans, A. actinomycetemcomitans plus adhesin and adhesin. MMP-8 activity increased with induction A. actinomycetemcomitans and 24 kDa A. actinomycetemcomitans adhesin. This fimbrial adhesin protein showed that A. actinomycetemcomitans has the ability to adhesion, colonization and invasion for host in aggressive periodontitis pathogenesis. Conclusion: A. actinomycetemcomitans fimbrial adhesin protein induction increasing MMP-8 activity for aggressive periodontitis pathogenesis.Latar belakang: A. actinomycetemcomitans merupakan salah satu bakteri Gram negatif yang terkait dengan periodontitis agresif yang menyerang penderita usia muda dan merupakan faktor penting dalam patogenesis periodontitis agresif. A. actimycetemcomitans mempunyai fimbriae dengan protein adhesin yang merupakan molekul pertama dari bakteri untuk melakukan kontak fisik dengan host. Tujuan: Tujuan penelitian ini adalah menganalisis pengaruh induksi adhesin A

  16. YadA, the multifaceted Yersinia adhesin.

    Science.gov (United States)

    El Tahir, Y; Skurnik, M

    2001-08-01

    The adhesion protein YadA is encoded by the yadA gene located in the 70-kb virulence plasmid of Yersinia (pYV) that is common to the pathogenic Yersinia species (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica). YadA is a virulence factor of Y. enterocolitica, however, YadA seems to be dispensable for the virulence of Y. pseudotuberculosis, and in wild-type Y. pestis the yadA gene has a frameshift mutation silencing the gene. Expression of the Y. pseudotuberculosis YadA in Y. pestis reduces its virulence. YadA is a homotrimer of ca. 45-kDa subunits that are anchored to the outer membrane via their C-termini, while their N-termini form a globular head on top of a stalk; the 'lollipop'-shaped YadA structure covers the entire bacterial surface giving it hydrophobic properties. The yadA gene expression is induced at 37 degrees C by the temperature-dependent transcriptional activator LcrF. YadA is a multifaceted protein as revealed by its different biological properties. YadA+ bacteria bind to collagens, laminin, fibronectin, intestinal submucosa, mucus, and to hydrophobic surfaces like polystyrene. YadA+ bacteria autoagglutinate in stationary culture and also specifically agglutinate guinea pig red blood cells. YadA is also a potent serum resistance factor as it inhibits the classical pathway of complement. As invasin, it mediates low rate invasion to tissue culture cells. In a rat model of reactive arthritis YadA and specifically YadA-mediated collagen binding is necessary for Y. enterocolitica to induce the disease. Despite of this wealth of information or perhaps because of it, the in vivo role of YadA during infection remains still largely unresolved.

  17. Identification and characterization of a novel Plasmodium falciparum adhesin involved in erythrocyte invasion.

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    Nidhi Hans

    Full Text Available Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.

  18. Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

    Science.gov (United States)

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Habermeyer, Johanna; Schwertz, Hansjörg; Hammerschmidt, Sven

    2017-04-07

    Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Importance of adhesins in the recurrence of pharyngeal infections caused by Streptococcus pyogenes.

    Science.gov (United States)

    Wozniak, Aniela; Scioscia, Natalia; Geoffroy, Enrique; Ponce, Iván; García, Patricia

    2017-04-01

    Pharyngo-amygdalitis is the most common infection caused by Streptococcus pyogenes (S. pyogenes). Reinfection with strains of different M types commonly occurs. However, a second infection with a strain of the same M type can still occur and is referred to as recurrence. We aimed to assess whether recurrence of S. pyogenes could be associated to erythromycin resistance, biofilm formation or surface adhesins like fibronectin-binding proteins and pilus proteins, both located in the fibronectin-binding, collagen-binding, T-antigen (FCT) region. We analyed clinical isolates of S. pyogenes obtained from children with multiple positive cultures of throat swabs. We analysed potential associations between M types, clonal patterns, biofilm production and FCT types with their capacity of producing a recurrent infection. We genetically defined recurrence as an infection with the same M type (same strain) and reinfection as an infection with a different M type. No differences were observed between recurrent and reinfection isolates in relation to erythromycin resistance, presence and number of domains of prtF1 gene, and biofilm formation capacity; the only significant difference was the higher frequency of FCT-4 type among recurrent isolates. However, when all the factors that could contribute to recurrence (erythromycin resistance, biofilm production, presence of prtF1 gene and FCT-4 type) were analysed together, we observed that recurrent isolates have a higher number of factors than reinfection isolates. Recurrence seems not to be associated with biofilm formation. However, pili and fibronectin-binding proteins could be associated with recurrence because FCT-4 isolates which harbour two fibronectin-binding proteins are more frequent among recurrent isolates.

  20. Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms.

    Science.gov (United States)

    Kraneveld, E A; de Soet, J J; Deng, D M; Dekker, H L; de Koster, C G; Klis, F M; Crielaard, W; de Groot, P W J

    2011-12-01

    An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

  1. Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum.

    Science.gov (United States)

    Guo, Lihong; Shokeen, Bhumika; He, Xuesong; Shi, Wenyuan; Lux, Renate

    2017-10-01

    Adhesin-mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species-specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non-mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram-positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD-SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species- and subspecies-dependent adhesin interactions. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Endocytosis-inducer adhesins produced by enteropathogenic serogroups of Escherichia coli participate on bacterial attachment to infant enterocytes

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    João Ramos Costa Andrade

    1987-03-01

    Full Text Available Enteropathogenic E. coli (EPEC infection of Hep-2 cells preoceeds through bacterial attachment to cell surface and internalization of adhered bacteria. EPEC attachment is a prerequisite for cell infection and is mediated by adhesins that recognize carbohydrate-containing receptors on cell membrane. Such endocytosis-inducer adhesins (EIA also promote EPEC binding to infant enterocytes, suggesting that EIA may have an important role on EPEC gastroenteritis.A infecção de células Hep-2 por E. coli enteropatogênicas (ECEP implica na aderência bacteriana e posterior interiorização dos microrganismos aderidos por um mecanismo de endocitose. A aderência das ECEP é pré-requisito para a infecção e é mediada por adesinas que reconhecem receptores inibidos por certas oses na membrana celular. Tais "adesinas indutoras da endocitose" (AIE também promovem a ligação bacteriana a enterócitos obtidos do intestino delgado de lactente, sugerindo que as AIE possam desempenhar algum papel nas diarréias causadas por ECEP.

  3. Structures of C-mannosylated anti-adhesives bound to the type 1 fimbrial FimH adhesin

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    Jerome de Ruyck

    2016-05-01

    Full Text Available Selective inhibitors of the type 1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against Escherichia coli infections such as urinary-tract infections. To construct these inhibitors, the α-d-mannopyranoside of high-mannose N-glycans, recognized with exclusive specificity on glycoprotein receptors by FimH, forms the basal structure. A hydrophobic aglycon is then linked to the mannose by the O1 oxygen inherently present in the α-anomeric configuration. Substitution of this O atom by a carbon introduces a C-glycosidic bond, which may enhance the therapeutic potential of such compounds owing to the inability of enzymes to degrade C-glycosidic bonds. Here, the first crystal structures of the E. coli FimH adhesin in complex with C-glycosidically linked mannopyranosides are presented. These findings explain the role of the spacer in positioning biphenyl ligands for interactions by means of aromatic stacking in the tyrosine gate of FimH and how the normally hydrated C-glycosidic link is tolerated. As these new compounds can bind FimH, it can be assumed that they have the potential to serve as potent new antagonists of FimH, paving the way for the design of a new family of anti-adhesive compounds against urinary-tract infections.

  4. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells

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    Hogan Robert J

    2010-09-01

    Full Text Available Abstract Background Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Results Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649 that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells and A549 (type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE. Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures. A second YadA-like gene product highly similar to BoaA (65% identity was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705. The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to

  5. Specificity of Campylobacter jejuni Adhesin PEB3 for Phosphates and Structural Differences among Its Ligand Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Min, Tongpil; Vedadi, Masoud; Watson, David C.; Wasney, Gregory A.; Munger, Christine; Cygler, Miroslaw; Matte, Allan; Young, N. Martin; (NRCC); (McGill); (Toronto)

    2009-04-22

    PEB3 is a glycoprotein adhesin from Campylobacter jejuni whose structure suggested a role in transport. We have investigated potential ligands for PEB3 and characterized their binding properties using biophysical methods in solution and by X-ray crystallography. A thermal aggregation assay of PEB3 with a library of physiological compounds identified three possible ligands [3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP), and aconitate], which stabilized wild-type PEB3 but did not stabilize either a PEB3 form containing two mutations at the ligand-binding site, T138A/S139A, or a second PEB3 mutant, K135E, at a site {approx}14 {angstrom} away. Fluorescence titration experiments and cocrystal structures with various ligands were used to characterize the binding of 3-PG, PEP, and phosphate to PEB3. Further, a C. jejuni growth experiment in minimal medium supplemented with 3-PG showed that this molecule enhances the growth of wild-type C. jejuni, but not of the PEB3 mutants. Crystallographic analysis of PEB3 complexes revealed that the Ser171-Gln180 region in the presence of 3-PG or other phosphates is helical and similar to those of other transport proteins, but it is nonhelical when citrate is bound. The K135E mutation resulted in expression of a more highly glycosylated form of PEB3 in vivo, and its crystal structure showed the conformation of the first two residues of the glycan. On the basis of our findings, we suggest that PEB3 is a transport protein that may function in utilization of 3-PG or other phosphate-containing molecules from the host.

  6. Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

    Science.gov (United States)

    Xiao, Longwen; Zhou, Liang; Sun, Changjiang; Feng, Xin; Du, ChongTao; Gao, Yu; Ji, Qun; Yang, Shuxin; Wang, Yu; Han, Wenyu; Langford, P R; Lei, Liancheng

    2012-10-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Essential roles and regulation of the Legionella pneumophila collagen-like adhesin during biofilm formation.

    Directory of Open Access Journals (Sweden)

    Julia Mallegol

    Full Text Available Legionellosis is mostly caused by Legionella pneumophila (Lp and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG-binding adhesin of Lp, named Lcl, is produced during legionellosis and is unique to the L. pneumophila species. Importantly, a mutant depleted in Lcl (Δlpg2644 is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the C-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS. In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of lpg2644, mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL, may play a role during the biofilm development. Thus, transcriptional down-regulation of lpg2644 may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.

  8. Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

    Science.gov (United States)

    Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

    2016-02-01

    Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors. Copyright © 2015. Published by Elsevier GmbH.

  9. Entamoeba histolytica: Adhesins and Lectins in the Trophozoite Surface

    Directory of Open Access Journals (Sweden)

    Magdalena Aguirre García

    2015-02-01

    Full Text Available Entamoeba histolytica is the causative agent of amebiasis in humans and is responsible for 100,000 deaths annually, making it the third leading cause of death due to a protozoan parasite. Pathogenesis appears to result from the potent cytotoxic activity of the parasite, which kills host cells within minutes. Although the mechanism is unknown, it is well established to be contact-dependent. The life cycle of the parasite alternates with two forms: the resistant cyst and the invasive trophozoite. The adhesive interactions between the parasite and surface glycoconjugates of host cells, as well as those lining the epithelia, are determinants for invasion of human tissues, for its cytotoxic activity, and finally for the outcome of the disease. In this review we present an overview of the information available on the amebic lectins and adhesins that are responsible of those adhesive interactions and we also refer to their effect on the host immune response. Finally, we present some concluding remarks and perspectives in the field.

  10. Oligomeric coiled-coil adhesin YadA is a double-edged sword.

    Directory of Open Access Journals (Sweden)

    Salome Casutt-Meyer

    Full Text Available Yersinia adhesin A (YadA is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Surprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadA(V₉₈D,N₉₉A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.

  11. Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery▿

    OpenAIRE

    Tsang, Tiffany M.; Felek, Suleyman; Krukonis, Eric S.

    2010-01-01

    Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, ...

  12. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

    Science.gov (United States)

    Ganguli, Kriston; Collado, Maria Carmen; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W. Allan; Rautava, Samuli

    2015-01-01

    Background Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Methods TNF-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Results Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. Conclusion The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human intestinal epithelial cells and directly modulates intestinal epithelial cell innate immune gene expression. PMID:25580735

  13. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

    Science.gov (United States)

    Ganguli, Kriston; Collado, Maria C; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W Allan; Rautava, Samuli

    2015-04-01

    Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

  14. Ail binding to fibronectin facilitates Yersinia pestis binding to host cells and Yop delivery.

    Science.gov (United States)

    Tsang, Tiffany M; Felek, Suleyman; Krukonis, Eric S

    2010-08-01

    Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Delta ail mutant had decreased binding to host cells, while a KIM5 Delta pla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Delta pla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.

  15. Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery▿

    Science.gov (United States)

    Tsang, Tiffany M.; Felek, Suleyman; Krukonis, Eric S.

    2010-01-01

    Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Δail mutant had decreased binding to host cells, while a KIM5 Δpla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Δpla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis. PMID:20498264

  16. MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Robinson, Mark W; Buchtmann, Kyle A; Jenkins, Cheryl; Tacchi, Jessica L; Raymond, Benjamin B A; To, Joyce; Roy Chowdhury, Piklu; Woolley, Lauren K; Labbate, Maurizio; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2013-04-17

    Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

  17. Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

    Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

  18. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii.

    Science.gov (United States)

    Lima, Bruno P; Shi, Wenyuan; Lux, Renate

    2017-06-01

    To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  19. Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms

    NARCIS (Netherlands)

    Kraneveld, E.A.; de Soet, J.J.; Deng, D.M.; Dekker, H.L.; de Koster, C.G.; Klis, F.M.; Crielaard, W.; de Groot, P.W.J.

    2011-01-01

    An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the

  20. The influence of adhesin protein from Aggregatibacter actinomycetemcomitans on IL-8 and MMP-8 titre in aggressive periodontitis

    Directory of Open Access Journals (Sweden)

    Rini Revijanti Ridwan

    2015-03-01

    Full Text Available Background: Adhesion can actually be considered as a part of both a powerful survival mechanism and a virulence mechanism for bacterial pathogens. Bacterial adhesin is an instrument for bacteria to do invasion to host. Bacterial adhesin depends on ligand interaction as a signaling mediator that will influence invasion and increase pro and anti-inflammatory because of the influence of the receptors of innate immune response. Aggregatibacter actimycetemcomitans has fimbriae included in type IV pili containing mostly with protein weighed 6.5 kDa and at least with protein weighed 54 kDa. Purpose: The purpose of this research is to analyze the influence of the induction of adhesin protein derived from A. actinomycetemcomitans on IL-8 and MMP-8 titre of Wistar rats. Methods: Adhesin protein derived from A. actinomycetemcomitans weighed 24 kDa was induced on the maxillary first molar sulcus of Wistar rats to prove that adhesin protein could affect IL-8 and MMP-8 titre. Next, to determine its influence, Elisa technique was conducted. Results: It is known that the levels of IL-8 and MMP-8 titre were increased in the group induced with adhesin protein derived from A. actinomycetemcomitans compared with the control group. Conclusion: It can be concluded that adhesin protein derived from A. actinomycetemcomitans can cause alveolar bone damage through the increasing levels of IL-8 and MMP-8 in aggressive periodontitis.

  1. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Goel, Suchi; Palmkvist, Mia; Moll, Kirsten

    2015-01-01

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum–encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs—preferentiall......Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum–encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs......—preferentially of blood group A—to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-18

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

  3. Genetic structure and expression of the surface glycoprotein GP82, the main adhesin of Trypanosoma cruzi metacyclic trypomastigotes.

    Science.gov (United States)

    Correa, Paulo Roberto Ceridorio; Cordero, Esteban Mauricio; Gentil, Luciana Girotto; Bayer-Santos, Ethel; da Silveira, José Franco

    2013-01-01

    T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. The metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. The function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.

  4. Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

    Energy Technology Data Exchange (ETDEWEB)

    Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori; Ikegami, Akihiko; Shoham, Menachem; Han, Yiping W.; (Case Western)

    2009-04-07

    Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.

  5. A Study of Surface Proteins, Other Adhesins and Iron Acquiring ...

    African Journals Online (AJOL)

    By the use guinea pig blood cells this was found to be 15% MRHA and 25% MSHA strains. When tested for their iron-binding protein (IBP) production, the MRHAs were positive for IBPs while the MSHA were positive for this property in 10%. Finally, based on the results obtained conclusions and recommendations are given ...

  6. Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua.

    Science.gov (United States)

    Thorell, Kaisa; Hosseini, Shaghayegh; Palacios Gonzáles, Reyna Victoria Palacios; Chaotham, Chatchai; Graham, David Y; Paszat, Lawrence; Rabeneck, Linda; Lundin, Samuel B; Nookaew, Intawat; Sjöling, Åsa

    2016-02-29

    Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. The complete genomes of fifty-two Nicaraguan H. pylori isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South- and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. The discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence properties.

  7. Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins.

    Science.gov (United States)

    Piñero-Lambea, Carlos; Bodelón, Gustavo; Fernández-Periáñez, Rodrigo; Cuesta, Angel M; Álvarez-Vallina, Luis; Fernández, Luis Ángel

    2015-04-17

    In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells. We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria.

  8. Comparison of adhesin genes and antimicrobial susceptibilities between uropathogenic and intestinal commensal Escherichia coli strains.

    Science.gov (United States)

    Qin, Xiaohua; Hu, Fupin; Wu, Shi; Ye, Xinyu; Zhu, Demei; Zhang, Ying; Wang, Minggui

    2013-01-01

    The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (PUPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (PUPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.

  9. Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis

    NARCIS (Netherlands)

    Que, Yok-Ai; Haefliger, Jacques-Antoine; Piroth, Lionel; François, Patrice; Widmer, Eleonora; Entenza, José M; Sinha, Bhanu; Herrmann, Mathias; Francioli, Patrick; Vaudaux, Pierre; Moreillon, Philippe

    2005-01-01

    The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected

  10. Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp

    OpenAIRE

    Engstrom, Michael D.; Mobley, Harry L. T.

    2016-01-01

    Urinary tract infections (UTIs) are a major burden to human health. The overwhelming majority of UTIs are caused by uropathogenic Escherichia coli (UPEC) strains. Unlike some pathogens, UPEC strains do not have a fixed core set of virulence and fitness factors but do have a variety of adhesins and regulatory pathways. One such UPEC adhesin is the nonfimbrial adhesin TosA, which mediates adherence to the epithelium of the upper urinary tract. The tos operon is AT rich, resides on pathogenicity...

  11. Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

    DEFF Research Database (Denmark)

    Schembri, Mark; Pallesen, Lars; Connell, Hugh

    1996-01-01

    on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58, and 279 of the mature FimH protein were shown to completely abolish binding to D......The gene encoding the Escherichia coli FimH adhesin has been subjected to linker insertion mutagenesis. Amino acid changes were introduced in a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations......-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding....

  12. Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

    Science.gov (United States)

    Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

    2015-01-01

    P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

  13. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    International Nuclear Information System (INIS)

    Nithianantham, Stanley; Xu, Minghua; Wu, Nan; Han, Yiping W.; Shoham, Menachem

    2006-01-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6 1 or P6 5 , and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6 1 or P6 5 , with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum

  14. Demarcating SurA Activities Required for Outer Membrane Targeting of Yersinia pseudotuberculosis Adhesins

    Science.gov (United States)

    Obi, Ikenna R.

    2013-01-01

    SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. We have previously reported that in enteropathogenic Yersinia pseudotuberculosis, SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization, and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting and a potentially compromised BAM complex assembly platform in the outer membrane. To various degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA, also depends on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process. PMID:23589578

  15. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    Energy Technology Data Exchange (ETDEWEB)

    Nithianantham, Stanley [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States); Xu, Minghua [Department of Biological Sciences, School of Dentistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4905 (United States); Wu, Nan [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States); Han, Yiping W., E-mail: ywh2@case.edu [Department of Biological Sciences, School of Dentistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4905 (United States); Department of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Shoham, Menachem, E-mail: ywh2@case.edu [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States)

    2006-12-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6{sub 1} or P6{sub 5}, and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.

  16. Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99.

    OpenAIRE

    Lee, J H; Isaacson, R E

    1995-01-01

    The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH. Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene. Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons. fanC was the most highly expressed, and fanD was expressed at very...

  17. Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

    Science.gov (United States)

    Bogema, Daniel R; Deutscher, Ania T; Woolley, Lauren K; Seymour, Lisa M; Raymond, Benjamin B A; Tacchi, Jessica L; Padula, Matthew P; Dixon, Nicholas E; Minion, F Chris; Jenkins, Cheryl; Walker, Mark J; Djordjevic, Steven P

    2012-01-01

    Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. Vaccines used to control Mycoplasma

  18. Surface contact stimulates the just-in-time deployment of bacterial adhesins.

    Science.gov (United States)

    Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

    2012-01-01

    The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive. © 2011 Blackwell Publishing Ltd.

  19. K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose

    Science.gov (United States)

    Sarabia-Sainz, Andre-i; Sarabia-Sainz, Hector Manuel; Ramos-Clamont Montfort, Gabriela; Mata-Haro, Veronica; Guzman-Partida, Ana María; Guzman, Roberto; Garcia-Soto, Mariano; Vazquez-Moreno, Luz

    2015-01-01

    The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-β (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10–17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections. PMID:26389896

  20. K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose

    Directory of Open Access Journals (Sweden)

    Andre-i Sarabia-Sainz

    2015-09-01

    Full Text Available The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88 was investigated. Glycated albumin with lactose (BSA-glucose-β (4-1 galactose was used as the microsphere matrix (MS-Lac and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3 were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10–17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections.

  1. Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae.

    Directory of Open Access Journals (Sweden)

    Preethi Ragunathan

    Full Text Available Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.

  2. Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA.

    Science.gov (United States)

    Nandre, Rahul M; Ruan, Xiaosai; Duan, Qiangde; Sack, David A; Zhang, Weiping

    2016-06-30

    Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    OpenAIRE

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription.

  4. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    Science.gov (United States)

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription. PMID:22595243

  5. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    Science.gov (United States)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  6. Structure of the Neisseria Adhesin Complex Protein (ACP) and its role as a novel lysozyme inhibitor.

    Science.gov (United States)

    Humbert, María Victoria; Awanye, Amaka Marian; Lian, Lu-Yun; Derrick, Jeremy P; Christodoulides, Myron

    2017-06-01

    Pathogenic and commensal Neisseria species produce an Adhesin Complex Protein, which was first characterised in Neisseria meningitidis (Nm) as a novel surface-exposed adhesin with vaccine potential. In the current study, the crystal structure of a recombinant (r)Nm-ACP Type I protein was determined to 1.4 Å resolution: the fold resembles an eight-stranded β-barrel, stabilized by a disulphide bond between the first (Cys38) and last (Cys121) β-strands. There are few main-chain hydrogen bonds linking β4-β5 and β8-β1, so the structure divides into two four-stranded anti-parallel β-sheets (β1-β4 and β5-β8). The computed surface electrostatic charge distribution showed that the β1-β4 sheet face is predominantly basic, whereas the β5-β8 sheet is apolar, apart from the loop between β4 and β5. Concentrations of rNm-ACP and rNeisseria gonorrhoeae-ACP proteins ≥0.25 μg/ml significantly inhibited by ~80-100% (Plysozyme (HL) over 24 h. Specificity was demonstrated by the ability of murine anti-Neisseria ACP sera to block ACP inhibition and restore HL activity. ACP expression conferred tolerance to HL activity, as demonstrated by significant 3-9 fold reductions (Plysozyme. In addition, wild-type Neisseria lactamica treated with purified ACP-specific rabbit IgG antibodies showed similar fold reductions in bacterial growth, compared with untreated bacteria (Pprotein family of lysozyme inhibitors. However, Neisseria ACP proteins show lysozyme recognition. These observations suggest that Neisseria ACP adopts a different mode of lysozyme inhibition and that the ability of ACP to inhibit lysozyme activity could be important for host colonization by both pathogenic and commensal Neisseria organisms. Thus, ACP represents a dual target for developing Neisseria vaccines and drugs to inhibit host-pathogen interactions.

  7. Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation.

    Science.gov (United States)

    Liu, Ping; Liu, Yi; Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

    2014-01-01

    Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (pgingivitis group than in health people (pgingivitis and periodontal disease compared with orthodontic gingivitis.

  8. Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis.

    Science.gov (United States)

    Rowe, Helen A; Griffiths, Natalie J; Hill, Darryl J; Virji, Mumtaz

    2007-01-01

    Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down-modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post-inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa-expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa-dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa-CEACAM binding substantially reduced cellular invasion, while maintaining

  9. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica

    Directory of Open Access Journals (Sweden)

    Arzu İrvem

    2016-10-01

    Full Text Available Background: E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol’s iodine for pre-diagnosis. Aims: In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Study Design: Retrospective cross-sectional study Methods: Preparations of stool samples stained with Native-Lugol’s iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson’s Chi-square and Fisher’s exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Results: Of 788 samples, 38 (4.8% were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01–6.330. Adhesin test-positivity was significant (p=0.047. Conclusion: In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were

  10. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica.

    Science.gov (United States)

    İrvem, Arzu; Özdil, Kamil; Çalışkan, Zuhal; Yücel, Muhterem

    2016-09-01

    E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol's iodine for pre-diagnosis. In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Retrospective cross-sectional study. Preparations of stool samples stained with Native-Lugol's iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson's Chi-square and Fisher's exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Of 788 samples, 38 (4.8%) were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01-6.330). Adhesin test-positivity was significant (p=0.047). In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were easily misjudged using direct microscopy. Although microscopic examination of samples

  11. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

    DEFF Research Database (Denmark)

    Pallesen, L; Poulsen, LK; Christiansen, Gunna

    1995-01-01

    of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing......The FimH adhesin of type 1 fimbriae has been tested as a display system for heterologous protein segments on the surface of Escherichia coli. This was carried out by introduction of restriction site handles (BglII sites) in two different positions in the fimH gene, followed by in-frame insertion...... as many as 56 foreign amino acids were transported to the bacterial surface as components of the fimbrial organelles. Furthermore, the foreign protein segments were recognized by insert-specific antibodies when expressed within chimeric proteins on the surface of the bacteria. The results from...

  12. The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

    Directory of Open Access Journals (Sweden)

    Lowe Anson W

    2009-07-01

    Full Text Available Abstract Background GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. Methods An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. Results GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Conclusion GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

  13. Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate.

    OpenAIRE

    Hacker, J; Kestler, H; Hoschützky, H; Jann, K; Lottspeich, F; Korhonen, T K

    1993-01-01

    S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Lik...

  14. Role of the inducible adhesin, CpAls7, in binding ofCandida parapsilosisto extracellular matrix under fluid shear.

    Science.gov (United States)

    Neale, Matthew N; Glass, Kyle A; Longley, Sarah J; Kim, Denny J; Laforce-Nesbitt, Sonia S; Wortzel, Jeremy D; Shaw, Sunil K; Bliss, Joseph M

    2018-01-29

    The yeast, Candida parapsilosis , is an increasingly common cause of systemic fungal infections among the immune compromised, including premature infants. Adhesion to host surfaces is an important step in pathogenesis, but this process has not been extensively studied in this organism. A microfluidics assay was developed to test the ability of C. parapsilosis to adhere to immobilized host extracellular matrix proteins under physiologic fluid shear conditions. Growth in mammalian tissue culture media at 37°C for 3-6 hours led to induction of an adhesive phenotype at shear forces of 1-5 dynes/cm 2 in some isolates of C. parapsilosis Glutamic acid, proline and calcium appeared to be the minimally necessary requirements for increased adhesion in these assays. To determine whether genes homologous to the ALS gene family of C. albicans were important for the adhesive phenotype, expression of 5 homologous C. parapsilosis genes were quantified using qPCR under conditions leading to increased adhesion. CPAR2_404800 ( CpALS7 ) and CPAR2_404780 showed increased expression compared to control yeast. The extent of adhesion was variable among different isolates, and linear regression identified expression of CpALS7 but not CPAR2_404780 to have a strong positive correlation with adhesion. A homozygous CpALS7 deletion strain was deficient in adhesion, whereas expression of CpALS7 in S. cerevisiae resulted in increased adhesion. Together, these data provide strong evidence that CpAls7 aids in the adherence of C. parapsilosis to extracellular matrix under shear forces and support its previously reported role in virulence. Copyright © 2018 American Society for Microbiology.

  15. Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis.

    Directory of Open Access Journals (Sweden)

    Kavindra V Singh

    2010-01-01

    Full Text Available Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001, while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace enhanced early (4 h heart valve colonization versus OG1RFDeltaace (pAT392 (P = 0.0418, suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce immunoglobulins (Igs, we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004 or sham-immunized (P = 0.0475 by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001 and showed fewer CFU in vegetations (P = 0.0146. In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

  16. Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

    Science.gov (United States)

    Vuong, Cuong; Kidder, Joshua B.; Jacobson, Erik R.; Otto, Michael; Proctor, Richard A.; Somerville, Greg A.

    2005-01-01

    Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production. PMID:15838022

  17. Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

    Science.gov (United States)

    Sillanpää, Jouko; Nallapareddy, Sreedhar R; Prakash, Vittal P; Qin, Xiang; Höök, Magnus; Weinstock, George M; Murray, Barbara E

    2008-10-01

    Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a

  18. The LapG protein plays a role in Pseudomonas aeruginosa biofilm formation by controlling the presence of the CdrA adhesin on the cell surface

    DEFF Research Database (Denmark)

    Rybtke, Morten; Berthelsen, Jens; Yang, Liang

    2015-01-01

    formation and biofilm dispersal. The P. aeruginosa LapG protein is shown to be a functional homolog of the Pseudomonas putida LapG protein which has previously been shown to function as a periplasmic protease that targets the surface adhesin LapA. Transposon mutagenesis and characterization of defined......Pseudomonas aeruginosa is a clinically relevant species involved in biofilm-based chronic infections. We provide evidence that the P. aeruginosa LapG protein functions as a periplasmic protease that can cleave the protein adhesin CdrA off the cell surface, and thereby plays a role in biofilm...

  19. Crystallization and X-ray diffraction analysis of a novel surface-adhesin protein: protein E from Haemophilus influenzae

    International Nuclear Information System (INIS)

    Singh, Birendra; Al Jubair, Tamim; Förnvik, Karolina; Thunnissen, Marjolein M.; Riesbeck, Kristian

    2012-01-01

    Protein E of the respiratory pathogen H. influenzae is a multifunctional adhesin that is involved in bacterial attachment to host epithelium and direct interactions with vitronectin, laminin and plasminogen. The method of crystallization and X-ray data collection for protein E at 1.8 Å is presented. Protein E (PE) is a ubiquitous multifunctional surface protein of Haemophilus spp. and other bacterial pathogens of the Pasteurellaceae family. H. influenzae utilizes PE for attachment to respiratory epithelial cells. In addition, PE interacts directly with plasminogen and the extracellular matrix (ECM) components vitronectin and laminin. Vitronectin is a complement regulator that inhibits the formation of the membrane-attack complex (MAC). PE-mediated vitronectin recruitment at the H. influenzae surface thus inhibits MAC and protects against serum bactericidal activity. Laminin is an abundant ECM protein and is present in the basement membrane that helps in adherence of H. influenzae during colonization. Here, the expression, purification and crystallization of and the collection of high-resolution data for this important H. influenzae adhesin are reported. To solve the phase problem for PE, Met residues were introduced and an SeMet variant was expressed and crystallized. Both native and SeMet-containing PE gave plate-like crystals in space group P2 1 , with unit-cell parameters a = 44, b = 57, c = 61 Å, β = 96°. Diffraction data collected from native and SeMet-derivative crystals extended to resolutions of 1.8 and 2.6 Å, respectively

  20. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

    Science.gov (United States)

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-01-01

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies. PMID:27387855

  1. Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

    Science.gov (United States)

    Lu, Yun-Yueh; Franz, Bettina; Truttmann, Matthias C; Riess, Tanja; Gay-Fraret, Jérémie; Faustmann, Marco; Kempf, Volkhard A J; Dehio, Christoph

    2013-05-01

    The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of

  2. Detection of pap, sfa, afa, foc, and fim Adhesin-Encoding Operons in Uropathogenic Escherichia coli Isolates Collected From Patients With Urinary Tract Infection.

    Science.gov (United States)

    Rahdar, Masoud; Rashki, Ahmad; Miri, Hamid Reza; Rashki Ghalehnoo, Mehdi

    2015-08-01

    Uropathogenic Escherichia coli (UPEC) with its virulence factors is the most prevalent cause of urinary tract infection (UTI). This study aimed to determine the occurrence of fim, pap, sfa, and afa genes among 100 UPEC isolates collected from patients diagnosed with UTI. A total of 100 UPEC isolates were obtained from urine samples of patients with UTI. The prevalence of 5 virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc) and afimbrial adhesins (afa) were determined through PCR method. We also investigated the phylogenetic background of all isolates. In addition, the distribution of adhesin-encoding operons between the phylogroups was assessed. The prevalence of genes encoding for fimbrial adhesive systems was 95% for fim, 57% for pap, 16% for foc, and 81% for sfa. The operons encoding for afa afimbrial adhesins were identified in 12% of isolates. The various combinations of detected genes were designated as virulence patterns. The fim gene, which occurred in strains from all phylogenetic groups (A, B1, B2, and D) was evaluated and no significant differences were found among these groups. Conversely, significant differences were observed in relation to pap, afa, foc, and sfa operons. These results indicate that the PCR method is a powerful genotypic assay for the detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as epidemiological studies.

  3. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

    NARCIS (Netherlands)

    Ganguli, K.; Collado, M.C.; Rautava, J.; Lu, L.; Satokari, R.M.; Ossowski, von I.; Reunanen, J.; Vos, de W.M.; Palva, A.; Isolauri, E.; Salminen, S.; Walker, W.A.; Rautava, S.

    2015-01-01

    BACKGROUND: Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human

  4. Ail Protein Binds Ninth Type III Fibronectin Repeat (9FNIII) within Central 120-kDa Region of Fibronectin to Facilitate Cell Binding by Yersinia pestis*

    Science.gov (United States)

    Tsang, Tiffany M.; Annis, Douglas S.; Kronshage, Malte; Fenno, Jesse T.; Usselman, Lisa D.; Mosher, Deane F.; Krukonis, Eric S.

    2012-01-01

    The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to 9FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of 9FNIII. Antibodies directed against 9FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats 9–10FNIII, and this binding was blocked by a mAb specific for 9FNIII. These data demonstrate that Ail binds to 9FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins. PMID:22447929

  5. Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis.

    Science.gov (United States)

    Tsang, Tiffany M; Annis, Douglas S; Kronshage, Malte; Fenno, Jesse T; Usselman, Lisa D; Mosher, Deane F; Krukonis, Eric S

    2012-05-11

    The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.

  6. The Yersinia pestis Ail protein mediates binding and Yop delivery to host cells required for plague virulence.

    Science.gov (United States)

    Felek, Suleyman; Krukonis, Eric S

    2009-02-01

    Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Deltaail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Deltaail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.

  7. Dimeric and trimeric fusion proteins generated with fimbrial adhesins of uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Víctor Manuel Luna-Pineda

    2016-10-01

    Full Text Available Urinary tract infections (UTIs are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion in the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc linked to the nucleotide sequence encoding the EAAAK5 peptide. Monomeric (fimH, csgA, and papG, dimeric (fimH-csgA, and trimeric (fimH-csgA-papG genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged fusion proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. The FimH, CsgA, and PapG stimulated the release of 372 to 398 pg/mL IL-6; interestingly, the FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018 and 521.24 pg/mL (p ≤ 0.002 IL-6, respectively. In addition, the FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001 and 450.40 pg/mL (p ≤ 0.002 IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit

  8. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of UropathogenicEscherichia coli.

    Science.gov (United States)

    Luna-Pineda, Víctor M; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH - csgA - papG - fimH - csgA ( fcpfc ) linked to the nucleotide sequence encoding the [EAAAK] 5 peptide. Monomeric ( fimH, csgA , and papG ), dimeric ( fimH-csgA ), and trimeric ( fimH - csgA - papG ) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL ( p ≤ 0.018) and 521.24 pg/mL ( p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL ( p ≤ 0.001) and 450.40 pg/mL ( p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine

  9. Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

    OpenAIRE

    Suits, Michael D.; Boraston, Alisdair B.

    2013-01-01

    PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-he...

  10. Plasmodium falciparum Adhesins Play an Essential Role in Signalling and Activation of Invasion into Human Erythrocytes.

    Directory of Open Access Journals (Sweden)

    Wai-Hong Tham

    2015-12-01

    Full Text Available The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL and reticulocyte binding-like (Rh protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process.

  11. Inhibition of Bifidobacterium Cell Wall 51.74 kDa Adhesin Isolated from Infants Feces Towards Adhesion of Enteric Phatogen E. coli on Enterocyte Balb/C Mice

    Directory of Open Access Journals (Sweden)

    I Sukrama

    2012-01-01

    Full Text Available Objectives: To determine 51.74 kDa adhesin of Bifidobacterium sp cell wall isolated from infants feces as an anti adhesion of E. coli on enterocyte mice. Methods: Randomized Posttest-Only Control Group Design was employed to investigate adherence ability of this adhesin towards E.coli adhesion on mice entherocyte. Results: In this research, it was obtained, that the 51.74 kDa adhesin cell wall of Bifidobacterium sp has an ability to inhibit adhesion of E. coli on mice enterocyte. The ability was increased as an increase of adhsein concentration. Conclusions: that can be drawn from this research is the finding of 51.74 kDa adhesin cell wall of Bifidobacterium sp isolated from infants feces that can inhibit adhseion of E. coli on mice enterocyte. Future work that can be carried out are further researches concerning whether these protein can be applied to inhibit adherence of other pathogen bacteria

  12. Salmonella enterica serotype Typhimurium Std fimbriae bind terminal alpha(1,2)fucose residues in the cecal mucosa.

    Science.gov (United States)

    Chessa, Daniela; Winter, Maria G; Jakomin, Marcello; Bäumler, Andreas J

    2009-02-01

    The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc) or by pretreatment of cells with alpha1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galbeta1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucalpha1-2Galbeta1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucalpha1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an alpha1-2 fucosylated receptor(s) present in the cecal mucosa.

  13. Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

    Science.gov (United States)

    Nandre, Rahul; Ruan, Xiaosai; Lu, Ti; Duan, Qiangde; Sack, David; Zhang, Weiping

    2018-03-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTa A14Q -dmLT or CFA/I/II/IV-2xSTa N12S -dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STa N12S , and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa + or LT + ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa + ETEC and LT + ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTa N12S -mnLT R192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin

  14. Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

    Science.gov (United States)

    2014-01-01

    SUMMARY The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as “silent pathogens” with the capacity to emerge as “pathobionts” for the development of inflammatory bowel disease and intestinal carcinogenesis. PMID:25278576

  15. Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

    Science.gov (United States)

    Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

  16. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Li, N.; Yun, P.; Nadkarni, M.A.; Ghadikolaee, N.B.; Nguyen, K.A.; Lee, M.; Hunter, N.; Collyer, C.A. (Sydney)

    2010-08-27

    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

  17. Vancomycin-Intermediate Staphylococcus aureus Strains Have Impaired Acetate Catabolism: Implications for Polysaccharide Intercellular Adhesin Synthesis and Autolysis▿

    Science.gov (United States)

    Nelson, Jennifer L.; Rice, Kelly C.; Slater, Sean R.; Fox, Paige M.; Archer, Gordon L.; Bayles, Kenneth W.; Fey, Paul D.; Kreiswirth, Barry N.; Somerville, Greg A.

    2007-01-01

    The most common mechanism by which Staphylococcus aureus gains resistance to vancomycin is by adapting its physiology and metabolism to permit growth in the presence of vancomycin. Several studies have examined the adaptive changes occurring during the transition to vancomycin-intermediate resistance, leading to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin. In the present study, we identified metabolic changes common to vancomycin-intermediate S. aureus (VISA) strains by assessing the metabolic and growth characteristics of two VISA strains (vancomycin MICs of 8 μg/ml) and two isogenic derivative strains with vancomycin MICs of 32 μg/ml. Interestingly, we observed the parental strains had impaired catabolism of nonpreferred carbon sources (i.e., acetate), and this impairment became more pronounced as vancomycin resistance increased. To determine if acetate catabolism impairment is common to VISA strains, we assessed the ability of VISA and vancomycin-sensitive S. aureus (VSSA) clinical isolates to catabolize acetate. As expected, a significantly greater percentage of VISA strains (71%) had impaired acetate catabolism relative to VSSA (8%). This is an important observation because staphylococcal acetate catabolism is implicated in growth yield and antibiotic tolerance and in regulating cell death and polysaccharide intercellular adhesin synthesis. PMID:17130298

  18. Trimeric autotransporter adhesins in members of the Burkholderia cepacia complex: a multifunctional family of proteins implicated in virulence

    Directory of Open Access Journals (Sweden)

    Arsénio Mendes Fialho

    2011-12-01

    Full Text Available Trimeric autotransporter adhesins (TAAs are multimeric surface proteins, involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains. In this study, a bioinformatic approach has been used to analyze the distribution and architecture of TAAs among Burkholderia cepacia complex (Bcc genomes. Fifteen genomes were probed revealing a total of 74 encoding sequences. Compared with other bacterial species, the Bcc genomes contain a disproportionately large number of TAAs (two genes to up to 8 genes, such as in B.cenocepacia. Phylogenetic analysis showed that the TAAs grouped into at least eight distinct clusters. TAAs with serine-rich repeats are clearly well separated from others, thereby representing a different evolutionary lineage. Comparative gene mapping across Bcc genomes reveals that TAA genes are inserted within conserved synteny blocks. We further focused our analysis on the epidemic strain B. cenocepacia J2315 in which 7 TAAs were annotated. Among these, 3 TAA-encoding genes (BCAM019, BCAM0223 and BCAM0224 are organized into a cluster and are candidates for multifunctional virulence factors. Here we review the current insights into the functional role of BCAM0224 as a model locus.

  19. Assessment of Pasteurella multocida A Lipopolysaccharide, as an Adhesin in an In Vitro Model of Rabbit Respiratory Epithelium

    Science.gov (United States)

    Romero, Stefany; Esquinas, Paula; Patiño, Pilar; Martínez, Nhora

    2017-01-01

    The role of the P. multocida lipopolysaccharide (LPS) as a putative adhesin during the early stages of infection with this bacterium in the respiratory epithelium of rabbits was investigated. By light microscopy and double enzyme labeling of nasal septa tissues, the amount of bacteria attached to the respiratory epithelium and the amount of LPS present in goblet cells at different experimental times were estimated. Transmission electron microscopy (TEM) and LPS labeling with colloidal gold particles were also used to determine the exact location of LPS in the cells. Septa that were challenged with LPS of P. multocida and 30 minutes later with P. multocida showed more adherent bacteria and more severe lesions than the other treatments. Free LPS was observed in the lumen of the nasal septum, forming bilamellar structures and adhering to the cilia, microvilli, cytoplasmic membrane, and cytoplasm of epithelial ciliated and goblet cells. The above findings suggest that P. multocida LPS plays an important role in the process of bacterial adhesion and that it has the ability of being internalized into host cells. PMID:28251016

  20. Staphylococcus epidermidis polysaccharide intercellular adhesin induces IL-8 expression in human astrocytes via a mechanism involving TLR2.

    LENUS (Irish Health Repository)

    Stevens, Niall T

    2009-03-01

    Staphylococcus epidermidis is an opportunistic biofilm-forming pathogen associated with neurosurgical device-related meningitis. Expression of the polysaccharide intercellular adhesin (PIA) on its surface promotes S. epidermidis biofilm formation. Here we investigated the pro-inflammatory properties of PIA against primary and transformed human astrocytes. PIA induced IL-8 expression in a dose- and\\/or time-dependent manner from U373 MG cells and primary normal human astrocytes. This effect was inhibited by depletion of N-acetyl-beta-d-glucosamine polymer from the PIA preparation with Lycopersicon esculentum lectin or sodium meta-periodate. Expression of dominant-negative versions of the TLR2 and TLR4 adaptor proteins MyD88 and Mal in U373 MG cells inhibited PIA-induced IL-8 production. Blocking IL-1 had no effect. PIA failed to induce IL-8 production from HEK293 cells stably expressing TLR4. However, in U373 MG cells which express TLR2, neutralization of TLR2 impaired PIA-induced IL-8 production. In addition to IL-8, PIA also induced expression of other cytokines from U373 MG cells including IL-6 and MCP-1. These data implicate PIA as an important immunogenic component of the S. epidermidis biofilm that can regulate pro-inflammatory cytokine production from human astrocytes, in part, via TLR2.

  1. The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins.

    Science.gov (United States)

    Shrivastava, Abhishek; Roland, Thibault; Berg, Howard C

    2016-09-06

    Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 μm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Assessment of Pasteurella multocida A Lipopolysaccharide, as an Adhesin in an In Vitro Model of Rabbit Respiratory Epithelium

    Directory of Open Access Journals (Sweden)

    Carolina Gallego

    2017-01-01

    Full Text Available The role of the P. multocida lipopolysaccharide (LPS as a putative adhesin during the early stages of infection with this bacterium in the respiratory epithelium of rabbits was investigated. By light microscopy and double enzyme labeling of nasal septa tissues, the amount of bacteria attached to the respiratory epithelium and the amount of LPS present in goblet cells at different experimental times were estimated. Transmission electron microscopy (TEM and LPS labeling with colloidal gold particles were also used to determine the exact location of LPS in the cells. Septa that were challenged with LPS of P. multocida and 30 minutes later with P. multocida showed more adherent bacteria and more severe lesions than the other treatments. Free LPS was observed in the lumen of the nasal septum, forming bilamellar structures and adhering to the cilia, microvilli, cytoplasmic membrane, and cytoplasm of epithelial ciliated and goblet cells. The above findings suggest that P. multocida LPS plays an important role in the process of bacterial adhesion and that it has the ability of being internalized into host cells.

  3. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

    Directory of Open Access Journals (Sweden)

    Devender Kumar

    2015-12-01

    Full Text Available Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

  4. Effect of simulated stages of the canine oestrous cycle on Escherichia coli binding to canine endometrium.

    Science.gov (United States)

    Krekeler, N; Lodge, K M; Anderson, G A; Browning, G F; Charles, J A; Wright, P J

    2012-12-01

    Pyometra, a prevalent infectious uterine disease that affects intact middle-aged bitches, is typically associated with Escherichia coli. Our hypotheses were (i) that bacterial adhesion to canine endometrium differs between different stages of the oestrous cycle and (ii) that the adhesin FimH facilitates this adhesion. Twelve post-pubertal, ovariectomized greyhound bitches were treated with exogenous hormones to simulate different stages of the oestrous cycle. Tissue samples from each uterus were incubated with a pathogenic E. coli strain carrying the fimH gene, but no other adhesin genes (P4-wt)--or an E. coli strain in which fimH was insertionally inactivated (P4-∆fimH::kan)--or with phosphate-buffered saline as a negative control. After washing, tissue samples were homogenized for quantification of adherent bacteria. The differences in binding to canine endometrium at different stages of the oestrous cycle were not significant. However, the mean difference in binding of the P4-wt and the P4-∆fimH::kan across all stages of the simulated oestrous cycle was significant (p < 0.001 by paired t-test on geometric means). Individual differences in numbers of P4-wt bacteria bound between dogs might suggest genetic variations or epigenetic differences in FimH receptor expression by the endometrium, unrelated to the stage of the oestrous cycle. © 2012 Blackwell Verlag GmbH.

  5. Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesin determinant: nucleotide sequence of the genes sfaB, C, D, E, F.

    OpenAIRE

    Schmoll, T.; Morschhäuser, J.; Ott, M.; Ludwig, B.; Van Die, I.; Hacker, Jörg

    2011-01-01

    The S fimbrial adhesin (sfa) determinant of E. co/i comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfaC situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation sturlies indicated th...

  6. Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF.

    Directory of Open Access Journals (Sweden)

    Andrio Lahesaare

    Full Text Available The biofilm matrix of the rhizospheric bacterium Pseudomonas putida consists mainly of a proteinaceous component. The two largest P. putida proteins, adhesins LapA and LapF, are involved in biofilm development but prevail in different developmental stages of the biofilm matrix. LapA is abundant in the initial stage of biofilm formation whereas LapF is found in the mature biofilm. Although the transcriptional regulation of the adhesins is not exhaustively studied, some factors that can be involved in their regulation have been described. For example, RpoS, the major stress response sigma factor, activates, and Fis represses LapF expression. This study focused on the LapF expression control by Fis. Indeed, using DNase I footprint analysis a Fis binding site Fis-F2 was located 150 bp upstream of the lapF gene coding sequence. The mapped 5' end of the lapF mRNA localized the promoter to the same region, overlapping with the Fis binding site Fis-F2. Monitoring the lapF promoter activity by a β-galactosidase assay revealed that Fis overexpression causes a 4-fold decrease in the transcriptional activity. Furthermore, mutations that diminished Fis binding to the Fis-F2 site abolished the repression of the lapF promoter. Thus, these data suggest that Fis is involved in the biofilm regulation via repression of LapF expression.

  7. Occurrence of genes of putative fibrinogen binding proteins and hemolysins, as well as of their phenotypic correlates in isolates of S. lugdunensis of different origins

    Directory of Open Access Journals (Sweden)

    Kaase Martin

    2011-04-01

    Full Text Available Abstract Background Staphylococcus lugdunensis is an important human pathogen that causes potentially fatal endocarditis, osteomyelitis and skin and soft tissue infections similar to diseases caused by Staphylococcus aureus. Nevertheless, in contrast to S. aureus, data on pathogenicity factors of S. lugdunensis is scarce. Two adhesins, a fibrinogen and a von Willebrand factor binding protein, and a S. lugdunensis synergistic hemolysin (SLUSH have been previously described. Moreover, the newly sequenced genome of S. lugdunensis revealed genes of other putative fibrinogen binding adhesins and hemolysins. The aim of this study was to gain more insight into the occurrence of genes likely coding for fibrinogen binding adhesins and hemolysins using clinical strains of S. lugdunensis. Findings Most of the putative adhesin genes and hemolysin genes investigated in this study were highly prevalent, except for the SLUSH gene cluster. In contrast to previous reports, binding to fibrinogen was detected in 29.3% of the S. lugdunensis strains. In most strains, hemolysis on blood agar plates was weak after 24 h and distinct after 48 h of incubation. The fibrinogen binding and hemolysis phenotypes were also independent of the type of clinical specimen, from which the isolates were obtained. Conclusion In this study we described a pyrrolidonyl arylamidase negative S. lugdunensis isolate. Our data indicate that a matrix-assisted laser desorption ionisation time-of-flight MS-based identification of S. lugdunensis or species-specific PCR's should be performed in favour of pyrrolidonyl arylamidase testing. In contrast to the high occurrence of putative fibrinogen binding protein genes, 29.3% of the S. lugdunensis strains bound to fibrinogen. Putative hemolysin genes were also prevalent in most of the S. lugdunensis strains, irrespective of their hemolysis activity on Columbia blood agar plates. Similar to a previous report, hemolysis after 48 h of incubation is also

  8. The Yersinia pestis Ail Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence▿

    Science.gov (United States)

    Felek, Suleyman; Krukonis, Eric S.

    2009-01-01

    Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Δail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Δail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice. PMID:19064637

  9. Expression, crystallization and preliminary X-ray data analysis of NT-Als9-2, a fungal adhesin from Candida albicans

    International Nuclear Information System (INIS)

    Salgado, Paula S.; Yan, Robert; Rowan, Fiona; Cota, Ernesto

    2011-01-01

    Details of the expression and crystallization of the N-terminal fragment of Als9-2, an adhesin from the human commensal/pathogenic fungus C. albicans, are reported. Preliminary analysis of the collected X-ray data is also discussed. Candida albicans is a common human fungal commensal that can also cause a range of infections from skin/mucosal ‘thrush’ to severe systemic candidiasis. Adherence to host cells is one of the key determinants of Candida pathogenesis. The Als family of surface proteins has been implicated in adhesion of C. albicans, yet limited information has been published on the structure and mechanism of these fungal adhesins. The N-terminal region of these proteins has been shown to possess adhesive properties, making it a possible target for new therapeutic strategies. Recombinant NT-Als9-2 from C. albicans (residues 18–329) was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.0 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 34.73, b = 68.71, c = 120.03 Å, α = β = γ = 90° and one molecule in the asymmetric unit. Platinum-derivatized crystals belonged to the same space group, with similar unit-cell parameters, although they were not completely isomorphous

  10. Identification of salivary mucin MUC7 binding proteins from Streptococcus gordonii

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    Thornton David J

    2009-08-01

    Full Text Available Abstract Background The salivary mucin MUC7 (previously known as MG2 can adhere to various strains of streptococci that are primary colonizers and predominant microorganisms of the oral cavity. Although there is a growing interest in interaction between oral pathogens and salivary mucins, studies reporting the specific binding sites on the bacteria are rather limited. Identification and characterization of the specific interacting proteins on the bacterial cell surface, termed adhesins, are crucial to further understand host-pathogen interactions. Results We demonstrate here, using purified MUC7 to overlay blots of SDS-extracts of Streptococcus gordonii cell surface proteins, 4 MUC7-binding bands, with apparent molecular masses of 62, 78, 84 and 133 kDa from the Streptococcus gordonii strain, PK488. Putative adhesins were identified by in-gel digestion and subsequent nanoLC-tandem mass spectrometry analysis of resultant peptides. The 62 kDa and 84 kDa bands were identified as elongation factor (EF Tu and EF-G respectively. The 78 kDa band was a hppA gene product; the 74 kDa oligopeptide-binding lipoprotein. The 133 kDa band contained two proteins; alpha enolase and DNA-directed RNA polymerase, beta' subunit. Some of these proteins, for example alpha enolase are expected to be intracellular, however, flow cytometric analysis confirmed its location on the bacterial surface. Conclusion Our data demonstrated that S. gordonii expressed a number of putative MUC7 recognizing proteins and these contribute to MUC7 mucin binding of this streptococcal strain.

  11. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.

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    Ho Seong Seo

    Full Text Available The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC, the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283-410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a "dock, lock, and latch" mechanism (DLL. Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.

  12. Alternate thermoregulation and functional binding of Escherichia coli type 1 fimbriae in environmental and animal isolates.

    Science.gov (United States)

    Marshall, Jacqueline; Rossez, Yannick; Mainda, Geoffrey; Gally, David L; Daniell, Tim J; Holden, Nicola J

    2016-11-01

    Type 1 fimbriae (T1F) are well characterised cell surface organelles expressed by Escherichia coli and required for adherence to mannosylated host tissue. They satisfy molecular Koch's postulates as a virulence determinant and a host-adapted role has been reinforced by reports that T1F expression is repressed at submammalian temperatures. Analysis of a group of 136 environmental and animal E. coli isolates that express T1F at 37°C showed that 28% are also capable of expression at 20°C, in a phase variable manner. The heterogeneous proportions varied widely, and although growth temperature impacted the total proportion expressing T1F, there was no direct correlation between growth at 37°C and 20°C, indicative of differences in thermoregulation of the genetic switch (fimS) that controls phase variation. Specificities of the adhesin (FimH) also varied between the isolates: most bound to α-(1-3) mannan and yeast extracts as expected, but some recognised β-(1-4)-mannans and N-linked glycoproteins from plants, and T1F from two of the isolates mediated binding to plant roots. The results expand our view of a well-described adherence factor to show alternative expression profiles and adhesin specificities, which in turn may confer an advantage for certain isolates in alternative hosts and habitats. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin.

    Science.gov (United States)

    Valeriano, Valerie Diane; Bagon, Bernadette B; Balolong, Marilen P; Kang, Dae-Kyung

    2016-07-01

    Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

  14. Identification of the psaA Gene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other than Streptococcus pneumoniae

    Science.gov (United States)

    Jado, Isabel; Fenoll, Asunción; Casal, Julio; Pérez, Amalia

    2001-01-01

    The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed. PMID:11527799

  15. Structural Context for Protein N-glycosylation in Bacteria: The Structure of PEB3, an Adhesin from Campylobacter Jejuni

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    Rangarajan,E.; Bhatia, S.; Watson, D.; Munger, C.; Cygler, M.; Matte, A.; Young, N.

    2007-01-01

    Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.

  16. Variability in the insect and plant adhesins, Mad1 and Mad2, within the fungal genus metarhizium suggest plant adaptation as an evolutionary force.

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    Michael Wyrebek

    Full Text Available Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2 or to insect association (Mad1 overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%, Mad1 open reading frame (6.7%, and Mad2 open reading frame (7.4% were similar, while it was higher in the Mad2 promoter region (9.9%. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1α, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1α than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

  17. Variability in the insect and plant adhesins, Mad1 and Mad2, within the fungal genus metarhizium suggest plant adaptation as an evolutionary force.

    Science.gov (United States)

    Wyrebek, Michael; Bidochka, Michael J

    2013-01-01

    Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1α, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1α than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

  18. Candida albicans Tpk1p and Tpk2p isoforms differentially regulate pseudohyphal development, biofilm structure, cell aggregation and adhesins expression.

    Science.gov (United States)

    Giacometti, Romina; Kronberg, Florencia; Biondi, Ricardo M; Passeron, Susana

    2011-04-01

    Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP-dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeast-to-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha:hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 Δ/tpk2 Δ strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 Δ/tpk2 Δ strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence. Copyright © 2011 John Wiley & Sons, Ltd.

  19. The Fibronectin-Binding Protein EfbA Contributes to Pathogenesis and Protects against Infective Endocarditis Caused by Enterococcus faecalis

    Science.gov (United States)

    Singh, Kavindra V.; La Rosa, Sabina Leanti; Somarajan, Sudha R.; Roh, Jung Hyeob

    2015-01-01

    EfbA is a PavA-like fibronectin adhesin of Enterococcus faecalis previously shown to be important in experimental urinary tract infection. Here, we expressed and purified the E. faecalis OG1RF EfbA and confirmed that this protein binds with high affinity to immobilized fibronectin, collagen I, and collagen V. We constructed an efbA deletion mutant and demonstrated that its virulence was significantly attenuated (P endocarditis model. Furthermore, efbA deletion resulted in diminished ability to bind fibronectin (P endocarditis (P = 0.008 versus control). Taken together, our results demonstrate that EfbA is an important factor involved in E. faecalis endocarditis and that rEfbA immunization is effective in preventing such infection, likely by interfering with bacterial adherence. PMID:26351286

  20. Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

    Science.gov (United States)

    Maurer, Lisa M; Tomasini-Johansson, Bianca R; Ma, Wenjiang; Annis, Douglas S; Eickstaedt, Nathan L; Ensenberger, Martin G; Satyshur, Kenneth A; Mosher, Deane F

    2010-12-24

    The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.

  1. Emerging ST121/agr4 community-associated methicillin-resistant Staphylococcus aureus (MRSA with strong adhesin and cytolytic activities: trigger for MRSA pneumonia and fatal aspiration pneumonia in an influenza-infected elderly

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    T.-W. Wan

    2016-09-01

    Full Text Available The pathogenesis of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA pneumonia in influenza-infected elderly individuals has not yet been elucidated in detail. In the present study, a 92-year-old man infected with influenza developed CA-MRSA pneumonia. His CA-MRSA was an emerging type, originated in ST121/agr4 S. aureus, with diversities of Panton–Valentine leucocidin (PVL−/spat5110/SCCmecV+ versus PVL+/spat159(etc./SCCmec−, but with common virulence potentials of strong adhesin and cytolytic activities. Resistance to erythromycin/clindamycin (inducible-type and gentamicin was detected. Pneumonia improved with the administration of levofloxacin, but with the subsequent development of fatal aspiration pneumonia. Hence, characteristic CA-MRSA with strong adhesin and cytolytic activities triggered influenza-related sequential complications.

  2. Prevalence of adhesin and toxin genes in E. coli strains isolated from diarrheic and non-diarrheic pigs from smallholder herds in northern and eastern Uganda.

    Science.gov (United States)

    Ikwap, Kokas; Larsson, Jenny; Jacobson, Magdalena; Owiny, David Okello; Nasinyama, George William; Nabukenya, Immaculate; Mattsson, Sigbrit; Aspan, Anna; Erume, Joseph

    2016-08-05

    Enterotoxigenic E. coli (ETEC) significantly contribute to diarrhea in piglets and weaners. The smallholder pig producers in Uganda identified diarrhea as one of the major problems especially in piglets. The aim of this study was to; i) characterize the virulence factors of E. coli strains isolated from diarrheic and non-diarrheic suckling piglets and weaners from smallholder herds in northern and eastern Uganda and ii) identify and describe the post-mortem picture of ETEC infection in severely diarrheic piglets. Rectal swab samples were collected from 83 piglets and weaners in 20 herds and isolated E. coli were characterized by PCR, serotyping and hemolysis. The E. coli strains carried genes for the heat stable toxins STa, STb and EAST1 and adhesins F4 and AIDA-I. The genes for the heat labile toxin LT and adhesins F5, F6, F18 and F41 were not detected in any of the E. coli isolates. Where the serogroup could be identified, E. coli isolates from the same diarrheic pig belonged to the same serogroup. The prevalence of EAST1, STb, Stx2e, STa, AIDA-I, and F4 in the E. coli isolates from suckling piglets and weaners (diarrheic and non-diarrheic combined) was 29, 26.5, 2.4, 1.2, 16, and 8.4 %, respectively. However the prevalence of F4 and AIDA-I in E. coli from diarrheic suckling piglets alone was 22.2 and 20 %, respectively. There was no significant difference in the prevalence of the individual virulence factors in E. coli from the diarrheic and non-diarrheic pigs (p > 0.05). The main ETEC strains isolated from diarrheic and non-diarrheic pigs included F4/STb/EAST1 (7.2 %), F4/STb (1.2 %), AIDA/STb/EAST1 (8 %) and AIDA/STb (8 %). At post-mortem, two diarrheic suckling piglets carrying ETEC showed intact intestinal villi, enterocytes and brush border but with a layer of cells attached to the brush border, suggestive of ETEC infections. This study has shown that the F4 fimbriae is the most predominant in E. coli from diarrheic piglets in the study area and

  3. Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium.

    Science.gov (United States)

    Krekeler, N; Marenda, M S; Browning, G F; Holden, K M; Charles, J A; Wright, P J

    2012-09-01

    Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

    Science.gov (United States)

    Nandakumar, Subhadra; Kannanganat, Sunil; Dobos, Karen M; Lucas, Megan; Spencer, John S; Fang, Sunan; McDonald, Melissa A; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V; Plikaytis, Bonnie B; Posey, James E; Amara, Rama Rao; Sable, Suraj B

    2013-01-01

    Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

  5. The Draft Genome of the Non-Host-Associated Methanobrevibacter arboriphilus Strain DH1 Encodes a Large Repertoire of Adhesin-Like Proteins

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    Anja Poehlein

    2017-01-01

    Full Text Available Methanobrevibacter arboriphilus strain DH1 is an autotrophic methanogen that was isolated from the wetwood of methane-emitting trees. This species has been of considerable interest for its unusual oxygen tolerance and has been studied as a model organism for more than four decades. Strain DH1 is closely related to other host-associated Methanobrevibacter species from intestinal tracts of animals and the rumen, making this strain an interesting candidate for comparative analysis to identify factors important for colonizing intestinal environments. Here, the genome sequence of M. arboriphilus strain DH1 is reported. The draft genome is composed of 2.445.031 bp with an average GC content of 25.44% and predicted to harbour 1964 protein-encoding genes. Among the predicted genes, there are also more than 50 putative genes for the so-called adhesin-like proteins (ALPs. The presence of ALP-encoding genes in the genome of this non-host-associated methanogen strongly suggests that target surfaces for ALPs other than host tissues also need to be considered as potential interaction partners. The high abundance of ALPs may also indicate that these types of proteins are more characteristic for specific phylogenetic groups of methanogens rather than being indicative for a particular environment the methanogens thrives in.

  6. The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates N-linked glycosylation of the Haemophilus influenzae HMW1 adhesin.

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    Kyoung-Jae Choi

    2010-12-01

    Full Text Available The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases.

  7. Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization.

    Science.gov (United States)

    Sakai, Takamitsu; Matsuyama, Tomomasa; Sano, Motohiko; Iida, Takaji

    2009-03-01

    Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.

  8. Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates.

    Science.gov (United States)

    Reissmann, Silvana; Gillen, Christine M; Fulde, Marcus; Bergmann, René; Nerlich, Andreas; Rajkumari, Reena; Brahmadathan, Kootallur N; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2012-01-01

    Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.

  9. A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells

    DEFF Research Database (Denmark)

    Claessens, Antoine; Adams, Yvonne; Ghumra, Ashfaq

    2012-01-01

    Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human...... of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0.......029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria....

  10. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  11. Ail proteins of Yersinia pestis and Y. pseudotuberculosis have different cell binding and invasion activities.

    Science.gov (United States)

    Tsang, Tiffany M; Wiese, Jeffrey S; Felek, Suleyman; Kronshage, Malte; Krukonis, Eric S

    2013-01-01

    The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ~50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30-50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.

  12. FimH adhesin of type 1 fimbriae is a potent inducer of innate antimicrobial responses which requires TLR4 and type 1 interferon signalling.

    Directory of Open Access Journals (Sweden)

    Ali A Ashkar

    2008-12-01

    Full Text Available Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs. We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-beta production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88(-/-, Trif(-/-, IFN-alpha/betaR(-/- or IRF3(-/- mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88(-/-, IFN-alpha/betaR(-/-, IRF3(-/- or TLR4(-/- mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-beta, but not IFN-alpha or IFN-gamma. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6 mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4(-/- mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.

  13. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  14. DNS & Bind Cookbook

    CERN Document Server

    Liu, Cricket

    2011-01-01

    The DNS & BIND Cookbook presents solutions to the many problems faced by network administrators responsible for a name server. Following O'Reilly's popular problem-and-solution cookbook format, this title is an indispensable companion to DNS & BIND, 4th Edition, the definitive guide to the critical task of name server administration. The cookbook contains dozens of code recipes showing solutions to everyday problems, ranging from simple questions, like, "How do I get BIND?" to more advanced topics like providing name service for IPv6 addresses. It's full of BIND configuration files that yo

  15. DNS BIND Server Configuration

    Directory of Open Access Journals (Sweden)

    Radu MARSANU

    2011-01-01

    Full Text Available After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  16. DNS BIND Server Configuratio

    OpenAIRE

    Radu MARSANU

    2011-01-01

    After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  17. DNS BIND Server Configuration

    OpenAIRE

    Radu MARSANU

    2011-01-01

    After a brief presentation of the DNS and BIND standard for Unix platforms, the paper presents an application which has a principal objective, the configuring of the DNS BIND 9 server. The general objectives of the application are presented, follow by the description of the details of designing the program.

  18. Melanin-binding radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  19. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    Packer, S.; Fairchild, R.G.; Watts, K.P.; Greenberg, D.; Hannon, S.J.

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed

  20. Functional mapping of YadA- and Ail-mediated binding of human factor H to Yersinia enterocolitica serotype O:3.

    Science.gov (United States)

    Biedzka-Sarek, Marta; Salmenlinna, Saara; Gruber, Markus; Lupas, Andrei N; Meri, Seppo; Skurnik, Mikael

    2008-11-01

    Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37 degrees C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding.

  1. Functional Mapping of YadA- and Ail-Mediated Binding of Human Factor H to Yersinia enterocolitica Serotype O:3▿

    Science.gov (United States)

    Biedzka-Sarek, Marta; Salmenlinna, Saara; Gruber, Markus; Lupas, Andrei N.; Meri, Seppo; Skurnik, Mikael

    2008-01-01

    Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37°C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding. PMID:18765735

  2. Decorin binding proteins of Borrelia burgdorferi promote arthritis development and joint specific post-treatment DNA persistence in mice.

    Directory of Open Access Journals (Sweden)

    Jemiina Salo

    Full Text Available Decorin binding proteins A and B (DbpA and B of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.

  3. The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

    Science.gov (United States)

    Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena

    2017-10-20

    The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

  4. Choline Binding Proteins from Streptococcus pneumoniae: A Dual Role as Enzybiotics and Targets for the Design of New Antimicrobials

    Directory of Open Access Journals (Sweden)

    Beatriz Maestro

    2016-06-01

    Full Text Available Streptococcus pneumoniae (pneumococcus is an important pathogen responsible for acute invasive and non-invasive infections such as meningitis, sepsis and otitis media, being the major cause of community-acquired pneumonia. The fight against pneumococcus is currently hampered both by insufficient vaccine coverage and by rising antimicrobial resistances to traditional antibiotics, making necessary the research on novel targets. Choline binding proteins (CBPs are a family of polypeptides found in pneumococcus and related species, as well as in some of their associated bacteriophages. They are characterized by a structural organization in two modules: a functional module (FM, and a choline-binding module (CBM that anchors the protein to the choline residues present in the cell wall through non-covalent interactions. Pneumococcal CBPs include cell wall hydrolases, adhesins and other virulence factors, all playing relevant physiological roles for bacterial viability and virulence. Moreover, many pneumococcal phages also make use of hydrolytic CBPs to fulfill their infectivity cycle. Consequently, CBPs may play a dual role for the development of novel antipneumococcal drugs, both as targets for inhibitors of their binding to the cell wall and as active cell lytic agents (enzybiotics. In this article, we review the current state of knowledge about host- and phage-encoded pneumococcal CBPs, with a special focus on structural issues, together with their perspectives for effective anti-infectious treatments.

  5. Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis.

    Science.gov (United States)

    Musyoki, Abednego Moki; Shi, Zhongyu; Xuan, Chunling; Lu, Guangwen; Qi, Jianxun; Gao, Feng; Zheng, Beiwen; Zhang, Qiangmin; Li, Yan; Haywood, Joel; Liu, Cuihua; Yan, Jinghua; Shi, Yi; Gao, George F

    2016-11-29

    The anchorless fibronectin-binding proteins (FnBPs) are a group of important virulence factors for which the structures are not available and the functions are not well defined. In this study we performed comprehensive studies on a prototypic member of this group: the fibronectin-/fibrinogen-binding protein from Streptococcus suis (FBPS). The structures of the N- and C-terminal halves (FBPS-N and FBPS-C), which together cover the full-length protein in sequence, were solved at a resolution of 2.1 and 2.6 Å, respectively, and each was found to be composed of two domains with unique folds. Furthermore, we have elucidated the organization of these domains by small-angle X-ray scattering. We further showed that the fibronectin-binding site is located in FBPS-C and that FBPS promotes the adherence of S suis to host cells by attaching the bacteria via FBPS-N. Finally, we demonstrated that FBPS functions both as an adhesin, promoting S suis attachment to host cells, and as a bacterial factor, activating signaling pathways via β1 integrin receptors to induce chemokine production.

  6. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  7. The Listeria monocytogenes LPXTG surface protein Lmo1413 is an invasin with capacity to bind mucin.

    Science.gov (United States)

    Mariscotti, Javier F; Quereda, Juan J; García-Del Portillo, Francisco; Pucciarelli, M Graciela

    2014-05-01

    Many Gram-positive bacterial pathogens use surface proteins covalently anchored to the peptidoglycan to cause disease. Bacteria of the genus Listeria have the largest number of surface proteins of this family. Every Listeria genome sequenced to date contains more than forty genes encoding surface proteins bearing anchoring-domains with an LPXTG motif that is recognized for covalent linkage to the peptidoglycan. About one-third of these proteins are present exclusively in pathogenic Listeria species, with some of them acting as adhesins or invasins that promote bacterial entry into eukaryotic cells. Here, we investigated two LPXTG surface proteins of the pathogen L. monocytogenes, Lmo1413 and Lmo2085, of unknown function and absent in non-pathogenic Listeria species. Lack of these two proteins does not affect bacterial adhesion or invasion of host cells using in vitro infection models. However, expression of Lmo1413 promotes entry of the non-invasive species L. innocua into non-phagocytic host cells, an effect not observed with Lmo2085. Moreover, overproduction of Lmo1413, but not Lmo2085, increases the invasion rate in non-phagocytic eukaryotic cells of an L. monocytogenes mutant deficient in the acting-binding protein ActA. Unexpectedly, production of full-length Lmo1413 and InlA exhibited opposite trends in a high percentage of L. monocytogenes isolates obtained from different sources. The idea of Lmo1413 playing a role as a new auxiliary invasin was also sustained by assays revealing that purified Lmo1413 binds to mucin via its MucBP domains. Taken together, these data indicate that Lmo1413, which we rename LmiA, for Listeria-mucin-binding invasin-A, may promote interaction of bacteria with adhesive host protective components and, in this manner, facilitate bacterial entry. Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature. The grids were subsequently .... and inhibition by GAGs and DMA were determined on polystyrene wells of microtitre plates (Costar, ... for binding inhibition assays was carried out by mixing equal volumes of the conjugate and the inhibitor at ...

  9. Quarkeosynthesis Binding Energy

    Science.gov (United States)

    Webb, Bill

    2009-05-01

    Quarkeosynthesis shows that the binding energy of a nucleus is the difference between the relativistic kinetic energies of its threesome of Jumbo Quarks and that of its building block quarks from neutrons and protons. There is no involvement of a nuclear strong force or gluon material.

  10. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  11. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity ... site is seen to correspond to the carbohydrate-binding site in E-selectin, which has similarity in the ... adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature.

  12. Molecular characterization of fibronectin-binding protein of ...

    African Journals Online (AJOL)

    The ability of lactobacilli to adhere to vaginal epithelial surfaces and intestinal tracts is believed to be important to allow colonization and host interaction for persistence. Bacterial adhesins molecules are proteins of which the human host fibronectin serves as a substrate for the attachment of bacteria and play a significant ...

  13. Technetium-99m labeling and fibronectin binding ability of Corynebacterium diphtheriae

    International Nuclear Information System (INIS)

    Souza, S.M.S.; Nagao, P.E.; Bernardo-Filho, M.; Pereira, G.A.; Napoleao, F.; Andrade, A.F.B.; Hirata Junior, R.; Mattos-Guaraldi, A.L.

    2004-01-01

    The use of radionuclides has permitted advances in areas of clinical and scientific knowledge. Several molecules and cells have been labelled with Technetium-99m ( 99m Tc). The stannous chloride (SnCl 2 ) has a significant influence on the labeling and stability of 99m Tc radiotracers. The frequent risk of diphtheria epidemics has intensified interest in the virulence factors of Corynebacterium diphtheriae. Although studies have looked at potential adhesins including haemagglutinins and exposed sugar residues, the molecular basis of mechanisms of adherence remains unclear. Adherence of pathogens to mammalian tissues may be mediated by fibronectin (FN) found in body fluids, matrix of connective tissues, and cell surfaces. In the present study we evaluated the binding ability to human plasma FN by 99m Tc labeled-C.diphtheriae. Due to adverse effects of stannous ions, microorganisms were submitted to survival and filamentation induction assays. Data showed a dose dependent susceptibility to SnCl 2 bactericidal effects. Cell filamentation was observed for concentrations of SnCl 2 > 110 μg/ml. Adherence levels of 99m Tc labelled 241strain to coverslips coated with 20 μg/ml FN were higher (P = 0.0037) than coated with bovine serum albumin. FN binding by the sucrose fermenting 241 C. diphtheriae strain (8.9% + 2.6) was significantly lower (P=0.0139) than Staphylococcus aureus Cowan I strain (34.1% ± 1.2). Therefore, bacterial 99m Tc labeling represents an additional tool that may contribute to the comprehension of C. diphtheriae interactions with host receptors such as FN that act as biological organizers by holding bacterial cells in position and guiding their migration. (author)

  14. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  15. Treponema pallidum subsp. pallidum TP0136 protein is heterogeneous among isolates and binds cellular and plasma fibronectin via its NH2-terminal end.

    Science.gov (United States)

    Ke, Wujian; Molini, Barbara J; Lukehart, Sheila A; Giacani, Lorenzo

    2015-03-01

    Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum (T. pallidum), the agent of syphilis. Among T. pallidum adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of T. pallidum binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein's central and COOH-terminal regions. Additionally, message quantification studies show that tp0136 is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in T. pallidum persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.

  16. Binding matrix: a novel approach for binding site recognition.

    Science.gov (United States)

    Kim, Jan T; Gewehr, Jan E; Martinetz, Thomas

    2004-06-01

    Recognition of protein-DNA binding sites in genomic sequences is a crucial step for discovering biological functions of genomic sequences. Explosive growth in availability of sequence information has resulted in a demand for binding site detection methods with high specificity. The motivation of the work presented here is to address this demand by a systematic approach based on Maximum Likelihood Estimation. A general framework is developed in which a large class of binding site detection methods can be described in a uniform and consistent way. Protein-DNA binding is determined by binding energy, which is an approximately linear function within the space of sequence words. All matrix based binding word detectors can be regarded as different linear classifiers which attempt to estimate the linear separation implied by the binding energy function. The standard approaches of consensus sequences and profile matrices are described using this framework. A maximum likelihood approach for determining this linear separation leads to a novel matrix type, called the binding matrix. The binding matrix is the most specific matrix based classifier which is consistent with the input set of known binding words. It achieves significant improvements in specificity compared to other matrices. This is demonstrated using 95 sets of experimentally determined binding words provided by the TRANSFAC database.

  17. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  18. Optical Binding of Nanowires

    Czech Academy of Sciences Publication Activity Database

    Simpson, Stephen Hugh; Zemánek, Pavel; Marago, O.M.; Jones, P.H.; Hanna, S.

    2017-01-01

    Roč. 17, č. 6 (2017), s. 3485-3492 ISSN 1530-6984 R&D Projects: GA ČR GB14-36681G Grant - others:AV ČR(CZ) CNR-16-12 Program:Bilaterální spolupráce Institutional support: RVO:68081731 Keywords : optical binding nanowires * Brownian motion * self -organization * non-equilibrium thermodynamics * non-equilibrium steady state * spin-orbit coupling * emergent phenomena Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 12.712, year: 2016

  19. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  20. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites.

    Science.gov (United States)

    Friedrich, Nikolas; Santos, Joana M; Liu, Yan; Palma, Angelina S; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

    2010-01-15

    Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for alpha2-3- over alpha2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to alpha2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6'sulfo-sialyl Lewis(x) might have implications for tissue tropism.

  1. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  2. Clonal distribution of bone sialoprotein-binding protein gene among Staphylococcus aureus isolates associated with bloodstream infections.

    Science.gov (United States)

    Wiśniewska, Katarzyna; Piórkowska, Anna; Kasprzyk, Joanna; Bronk, Marek; Świeć, Krystyna

    2014-11-01

    Staphylococcus aureus is a leading cause of bloodstream infections (BSI) and diseases that may be caused by hematogenous spread. The staphylococcal adhesin, for which the association with the infections emerging as a complication of septicemia has been well documented, is a bone sialoprotein-binding protein (Bbp). The aim of the study was to assess the prevalence of a bbp gene in S. aureus bloodstream isolates associated with BSI and to investigate to what degree the distribution of this gene is linked to the clonality of the population. Spa typing, used in order to explore the genetic population structure of the isolates, yielded 29 types. Six spa clusters and seven singletons were identified. The most frequent was spa clonal complex CC021 associated with MLST CC30 (38%). The bbp gene was found in 47% of isolates. Almost all isolates (95%) clustered in spa clonal complex CC021 were positive for this gene. All isolates carrying the bbp gene were sensitive to methicillin, and if clustered in the spa CC021, belonged to agr group III. Our study shows that Bbp is not strictly associated with BSI. However, one may conclude that for clonally related S. aureus strains most commonly causing BSI, the risk of Bbp-mediated complications of septicemia is expected to be higher than for other strains.

  3. The PavA-like fibronectin-binding protein of Enterococcus faecalis, EfbA, is important for virulence in a mouse model of ascending urinary tract infection.

    Science.gov (United States)

    Torelli, Riccardo; Serror, Pascale; Bugli, Francesca; Paroni Sterbini, Francesco; Florio, Ada Rita; Stringaro, Annarita; Colone, Marisa; De Carolis, Elena; Martini, Cecilia; Giard, Jean-Christophe; Sanguinetti, Maurizio; Posteraro, Brunella

    2012-09-15

    Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.

  4. Pictorial binding: endeavor to classify

    Directory of Open Access Journals (Sweden)

    Zinchenko S.

    2015-01-01

    Full Text Available The article is devoted to the classification of bindings of the 1-19th centuries with a unique and untypical book binding decoration technique (encaustic, tempera and oil paintings. Analysis of design features, materials and techniques of art decoration made it possible to identify them as a separate type - pictorial bindings and divide them into four groups. The first group consists of Coptic bindings, decorated with icon-painting images in encaustic technique. The second group is made up of leather Western bindings of the 13-14th centuries, which have the decoration and technique of ornamentation close to iconography. The third group involves parchment bindings, ornamentation technique of which is closer to the miniature. The last group comprises bindings of East Slavic origin of the 15-19th centuries, decorated with icon-painting pictures made in the technique of tempera or oil painting. The proposed classification requires further basic research as several specific kinds of bindings have not yet been investigated

  5. Metal binding by food components

    DEFF Research Database (Denmark)

    Tang, Ning

    For calcium binding: Electrochemical method (calcium ion selective electrode) combined with quantum mechanical calculations (density functional theory) were used to investigate the calcium binding affinity of the amino acids and small glycine peptides. The effects of the ionic strength and p......, synergistic effect in calcium binding was found for the small glycine peptide rather than amino acids mixtures with the enhanced driving force up to -6 kJ/mol. Such study provides useful information for the future development of calcium supplements. For zinc binding: Isothermal titration calorimetry...... titration calorimetry and quantum mechanical calculations. This is due to the zinc binding affinity of the relatively softer ligands (investigated food components) will become much stronger than citrate or phytate when they present together in aqueous solution. This mechanism indicates these food components...

  6. Host iron binding proteins acting as niche indicators for Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Philip W Jordan

    Full Text Available Neisseria meningitidis requires iron, and in the absence of iron alters its gene expression to increase iron acquisition and to make the best use of the iron it has. During different stages of colonization and infection available iron sources differ, particularly the host iron-binding proteins haemoglobin, transferrin, and lactoferrin. This study compared the transcriptional responses of N. meningitidis, when grown in the presence of these iron donors and ferric iron, using microarrays.Specific transcriptional responses to the different iron sources were observed, including genes that are not part of the response to iron restriction. Comparisons between growth on haemoglobin and either transferrin or lactoferrin identified changes in 124 and 114 genes, respectively, and 33 genes differed between growth on transferrin or lactoferrin. Comparison of gene expression from growth on haemoglobin or ferric iron showed that transcription is also affected by the entry of either haem or ferric iron into the cytoplasm. This is consistent with a model in which N. meningitidis uses the relative availability of host iron donor proteins as niche indicators.Growth in the presence of haemoglobin is associated with a response likely to be adaptive to survival within the bloodstream, which is supported by serum killing assays that indicate growth on haemoglobin significantly increases survival, and the response to lactoferrin is associated with increased expression of epithelial cell adhesins and oxidative stress response molecules. The transferrin receptor is the most highly transcribed receptor and has the fewest genes specifically induced in its presence, suggesting this is the favoured iron source for the bacterium. Most strikingly, the responses to haemoglobin, which is associated with unrestricted growth, indicates a low iron transcriptional profile, associated with an aggressive phenotype that may be adaptive to access host iron sources but which may also

  7. Pro DNS and BIND 10

    CERN Document Server

    Aitchison, Ron

    2011-01-01

    Pro DNS and BIND 10 guides you through the challenging array of features surrounding DNS with a special focus on the latest release of BIND, the world's most popular DNS implementation. This book unravels the mysteries of DNS, offering insight into origins, evolution, and key concepts like domain names and zone files. This book focuses on running DNS systems based on BIND 10, the first stable release that includes support for the latest DNSSEC standards. Whether you administer a DNS system, are thinking about running one, or you simply want to understand the DNS system, then this book for you.

  8. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  9. Boiling eXperiment Facility (BXF) Fluid Toxicity Technical Interchange Meeting (TIM) with the Payload Safety Review Panel (PSRP)

    Science.gov (United States)

    Sheredy, William A.

    2012-01-01

    A Technical Interchange meeting was held between the payload developers for the Boiling eXperiment Facility (BXF) and the NASA Safety Review Panel concerning operational anomaly that resulted in overheating one of the fluid heaters, shorted a 24VDC power supply and generated Perfluoroisobutylene (PFiB) from Perfluorohexane.

  10. Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus.

    Directory of Open Access Journals (Sweden)

    Lifang Qi

    Full Text Available Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3, were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04-14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide, 20.2 ± 0.5 (P3, 5.1 ± 0.7 (P1 and 3.4 ± 0.8 (P2, respectively. There was a significant decrease in the adhesive level of 04-14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01. The affinity constants of P1 and P2 were (6.17 ± 0.19 × 108 L/mol and (1.24 ± 0.56 × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

  11. Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus.

    Science.gov (United States)

    Qi, Lifang; Liu, Yan; Tao, Huizhu; Xiao, Ning; Li, Jinnian; Kong, Lingyan; Hou, Liting

    2016-01-01

    Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04-14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04-14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

  12. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... and Iron-binding Capacity (TIBC, UIBC) Trichomonas Testing Triglycerides Troponin Tryptase Tumor Markers Uric Acid Urinalysis Urine ... Syndrome CME. Medscape From Nature Clinical Practice Endocrinology & Metabolism [On-line information]. Available online at http://www. ...

  13. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...... and KCN, are selectively bound to the catalyst, providing exceptionally high enantioselectivities for kinetic resolutions, elimination reactions (fluoride base), and Strecker synthesis (cyanide nucleophile). Asymmetric cation-binding catalysis was recently expanded to silicon-based reagents, enabling...

  14. Some adhesins of avian pathogenic Escherichia coli (APEC isolated from septicemic poultry in Brazil Algumas adesinas de Escherichia coli aviária (APEC isoladas de aves com colisepticemia no Brasil

    Directory of Open Access Journals (Sweden)

    Terezinha Knöbl

    2006-09-01

    Full Text Available Three hundred and fifty strains of E. coli isolated from septicemic poultry from seven states of Brazil were examined for presence of nine adhesion-encoding genes, hemagglutination and adherence to chicken tracheal cells (in vitro. Analysis of the strains by colony hybridization tests demonstrated that 93.7% of the isolates were fim +, 17% pap+ and 5.7% were sfa+. The mannose sensitive fimbriae occur with similar frequency in APEC isolated from all Brazilians states, while significant differences among pap and sfa genes distributions were observed. The results showed that 0.85% and 0.28% of APEC were positive for genes that encoded enteroaggregative adhesins and EPEC adherence factor, respectively. None of APEC was positive for DA, afa, Bfp and Eae probes. The adherence to chicken tracheal cells showed 96% positive strains, while hemagglutination assays showed 26.5% of the isolates were mannose sensitive and 21.7% were mannose resistant.Trezentas e cinqüenta amostras de E. coli isoladas de aves com septicemia em sete estados do Brasil foram examinadas para a presença de nove genes codificadores de adesinas, hemaglutinação e aderência em células da traquéia (in vitro. A análise das amostras pela hibridização de colônias demonstrou que 93,7% dos isolados eram fim +, 17% pap+ e 5,7% eram sfa+. As fímbrias manose sensíveis apresentaram uma distribuição uniforme em todos os estados do Brasil. No entanto, diferenças significativas na distribuição dos genes pap e sfa foram observadas. Os resultados mostraram que 0,85% e 0,28% das APEC foram positivas para os genes que codificam as adesinas enteroagregativas e o fator de aderência de EPEC, respectivamente. Nenhuma amostra foi positiva para as sondas DA, afa, Bfp e Eae. A aderência em células de traquéia de aves revelou 96% de amostras positivas, enquanto os testes de hemaglutinação mostraram 26,5% dos isolados mannose sensíveis e 21,7% manose resistentes.

  15. Water binding in legume seeds

    Science.gov (United States)

    Vertucci, C. W.; Leopold, A. C.

    1987-01-01

    The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

  16. Structure of a Streptococcal Adhesin Carbohydrate Receptor

    Science.gov (United States)

    1990-08-01

    correlation spectroscopy; PMAA, partially methylated alditol Research, Washington, DC 20307. Tel.: 202-576-2582. acetates; FAB, fast atom bombardment. This...Analysis of the partially methylated alditol acetate derivatives subunit linked by phosphodiester bonds (17). The c-oaggrega- was performed on an HP5890/5970...34opposite" by virtue of the mass spectra and retention times of the direction, as follows: resulting partially methylated alditol acetates (PMAA

  17. Characterization of Helicobacter pylori adhesin thiol peroxidase ...

    Indian Academy of Sciences (India)

    Prakash

    , pH 7.0. The purified protein was detected mainly in the flow-through and its purity was analysed by. SDS-PAGE. The protein sample was then concentrated and stored at –80°C for ... buffer, pH 7.0 in the presence or absence of 10 mM DTT for.

  18. Fimbrial adhesins from extraintestinal Escherichia coli

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

  19. Molecular design of Mycoplasma hominis Vaa adhesin

    DEFF Research Database (Denmark)

    Boesen, Thomas; Fedosova, Natalya U.; Kjeldgaard, Morten

    2001-01-01

    distinct coiled-coil regions. The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two-cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating...... by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80%. Sequence analysis indicates the presence...

  20. Characterization of Helicobacter pylori adhesin thiol peroxidase ...

    Indian Academy of Sciences (India)

    Prakash

    DED plus HpTpx. 3. Results and discussion. 3.1 Purification of recombinant HpTpx protein. Recombinant His6-HpTpx protein was overexpressed in. E. coli and purified to homogeneity from lysates using nickel affinity chromatography followed ...

  1. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  2. Calcium binding by dietary fibre

    International Nuclear Information System (INIS)

    James, W.P.T.; Branch, W.J.; Southgate, D.A.T.

    1978-01-01

    Dietary fibre from plants low in phytate bound calcium in proportion to its uronic-acid content. This binding by the non-cellulosic fraction of fibre reduces the availability of calcium for small-intestinal absorption, but the colonic microbial digestion of uronic acids liberates the calcium. Thus the ability to maintain calcium balance on high-fibre diets may depend on the adaptive capacity on the colon for calcium. (author)

  3. Ligand binding by PDZ domains

    DEFF Research Database (Denmark)

    Chi, Celestine N.; Bach, Anders; Strømgaard, Kristian

    2012-01-01

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common protein-protein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins...... as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context....

  4. Binding energy of protonium ions

    Science.gov (United States)

    Assad Abdel-Raouf, Mohamed

    2009-11-01

    The goal of the present work is to calculate the binding energy of the protonium ions bar PPe+ and bar PPe- using Rayleigh- Ritz variational method. It is indicated that an employment of 21 components of the trial wavefunction yields -0.08793 eV as the ground state energy of these ions. Our result agrees quite well with recently obtained results based on elaborate Monte Carlo approximations. It confirms the possible formation of these ions in laboratory.

  5. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

  6. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  7. Anion binding in biological systems

    International Nuclear Information System (INIS)

    Feiters, Martin C; Meyer-Klaucke, Wolfram; Kostenko, Alexander V; Soldatov, Alexander V; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Kuepper, Frithjof C; Hollenstein, Kaspar; Locher, Kaspar P; Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2009-01-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L 3 (2p 3/2 ) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  8. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  9. A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

    Directory of Open Access Journals (Sweden)

    Reeja Maria Cherian

    2015-08-01

    Full Text Available Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b, CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1, core 3 (B3GNT6, core 4 (GCNT1 and B3GNT6, or extended core 1 (B3GNT3 chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc or type 2 (Galb4GlcNAc outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

  10. Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells*

    Science.gov (United States)

    Meyrand, Mickael; Guillot, Alain; Goin, Mélodie; Furlan, Sylviane; Armalyte, Julija; Kulakauskas, Saulius; Cortes-Perez, Naima G.; Thomas, Ginette; Chat, Sophie; Péchoux, Christine; Dupres, Vincent; Hols, Pascal; Dufrêne, Yves F.; Trugnan, Germain; Chapot-Chartier, Marie-Pierre

    2013-01-01

    Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells

  11. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    Wolverton, C.

    2007-01-01

    Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

  12. Lactoferrin binding molecules in human seminal plasma.

    Science.gov (United States)

    Thaler, C J; Vanderpuye, O A; McIntyre, J A; Faulk, W P

    1990-10-01

    During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.

  13. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein a...

  14. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  15. Drug binding properties of neonatal albumin

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B

    1989-01-01

    Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed...... by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug is bound...... per albumin molecule, and binding of the first sulfamethizole molecule is possibly reduced as well. Diazepam binds with equal affinity to the fetal and adult proteins. Among the two main albumin drug-binding functions, for warfarin and diazepam, the former is thus compromised in the newborn infant...

  16. Insulin binding to individual rat skeletal muscles

    International Nuclear Information System (INIS)

    Koerker, D.J.; Sweet, I.R.; Baskin, D.G.

    1990-01-01

    Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white [extensor digitorum longus (EDL), gastrocnemius] muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding

  17. Insulin binding to individual rat skeletal muscles

    Energy Technology Data Exchange (ETDEWEB)

    Koerker, D.J.; Sweet, I.R.; Baskin, D.G. (Univ. of Washington, Seattle (USA))

    1990-10-01

    Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white (extensor digitorum longus (EDL), gastrocnemius) muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding.

  18. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...... diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract...

  19. Binding characteristics of swine erythrocyte insulin receptors

    Energy Technology Data Exchange (ETDEWEB)

    Dieberg, G.; Bryan, G.S.; Sartin, J.L.; Williams, J.C.; Prince, T.J.; Kemppainen, R.J.

    1985-09-01

    Crossbred gilts had 8.8 +/- 1.1% maximum binding of ( SVI)insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows had a significant increase in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet and a low-fiber diet did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine.

  20. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  1. Synthetic LPS-Binding Polymer Nanoparticles

    Science.gov (United States)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  2. Sex hormone binding globulin phenotypes

    DEFF Research Database (Denmark)

    Cornelisse, M M; Bennett, Patrick; Christiansen, M

    1994-01-01

    Human sex hormone binding globulin (SHBG) is encoded by a normal and a variant allele. The resulting SHBG phenotypes (the homozygous normal SHBG, the heterozygous SHBG and the homozygous variant SHBG phenotype) can be distinguished by their electrophoretic patterns. We developed a novel detection....... This method of detection was used to determine the distribution of SHBG phenotypes in healthy controls of both sexes and in five different pathological conditions characterized by changes in the SHBG level or endocrine disturbances (malignant and benign ovarian neoplasms, hirsutism, liver cirrhosis...... on the experimental values. Differences in SHBG phenotypes do not appear to have any clinical significance and no sex difference was found in the SHBG phenotype distribution....

  3. Binding thermodynamics of a glutamate transporter homologue

    Science.gov (United States)

    Reyes, Nicolas; Oh, SeCheol; Boudker, Olga

    2013-01-01

    Glutamate transporters catalyze concentrative uptake of the neurotransmitter into glial cells and neurons. Their transport cycle involves binding and release of the substrate on the extra- and intracellular sides of the plasma membranes, and translocation of the substrate-binding site across the lipid bilayers. The energy of the ionic gradients, mainly sodium, fuels the cycle. Here, we used a cross-linking approach to trap a glutamate transporter homologue from Pyrococcus horikoshii in key conformational states with substrate-binding site facing either the extracellular or intracellular sides of the membrane to study their binding thermodynamics. We show that the chemical potential of sodium ions in solution is exclusively coupled to substrate binding and release, and not to substrate translocation. Despite the structural symmetry, the binding mechanisms are distinct on the opposite sides of the membrane and more complex than the current models suggest. PMID:23563139

  4. Characterization of feline serum-cobalt binding.

    Science.gov (United States)

    Schnelle, Amy N; Barger, Anne M; MacNeill, Amy L; Mitchell, Mark M; Solter, Philip

    2015-06-01

    Oxidative stress inhibits albumin's ability to complex with cobalt. Feline serum-cobalt binding has not been described. The objective was to develop a cobalt binding test for use with feline serum, and correlate the results with other biochemical and cellular constituents in blood, and with clinical diseases of cats. A colorimetric test of cobalt binding, based on the oxidation-reduction reaction of Co(+2) and dithiothreitol, was developed using feline serum. The test was used to measure cobalt binding in stored serum from 176 cats presented to the University of Illinois Veterinary Teaching Hospital for a variety of disease conditions. Time-matched hematology and biochemical data, and clinical information, were obtained from the medical record of each cat and correlated with the serum-cobalt binding results. Serial dilution of feline serum with phosphate-buffered saline resulted in a highly linear decrease in serum-cobalt binding (r(2)  = .9984). Serum-cobalt binding of the clinical samples also correlated with albumin concentrations in a stepwise linear regression model (r(2)  = .425), and both cobalt binding and albumin were significantly decreased in cases of inflammation. Albumin and cobalt binding also shared significant correlations with several erythron variables, and serum concentration of total calcium and bilirubin. The correlation of cobalt binding measured by a colorimetric test with albumin concentration in the clinical samples and with serum dilution is consistent with feline albumin-cobalt complex formation. Hypoalbuminemia is the likely cause of reduced serum-cobalt binding in inflammation and the correlations observed between cobalt binding and other variables. © 2015 American Society for Veterinary Clinical Pathology.

  5. Binding energy of the barbell exciton

    Science.gov (United States)

    Peeters, F. M.; Golub, J. E.

    1991-02-01

    The exciton binding energy in asymmetric coupled double quantum wells is calculated. As the system is electrically tuned from type I to type II, the exciton binding energy decreases from that of a two-dimensional exciton to the binding energy of a spatially separated electron-hole pair, i.e., the barbell exciton.$-- We compare our theoretical results with a recent experiment and find good agreement.

  6. CAP binding proteins associated with the nucleus.

    OpenAIRE

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in in...

  7. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin.

    Directory of Open Access Journals (Sweden)

    Hirohito Abo

    Full Text Available We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA, revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG, heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.

  8. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin.

    Science.gov (United States)

    Abo, Hirohito; Soga, Keisuke; Tanaka, Atsuhiro; Tateno, Hiroaki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.

  9. Drug binding properties of neonatal albumin

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B

    1989-01-01

    Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed...... by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug is bound...

  10. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  11. Parathyroid hormone binding to cultured avian osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Teti, A.; Rizzoli, R.; Zambonin Zallone, A. (Univ. of Bari (Italy))

    1991-02-14

    Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with ({sup 125}I)--bovine PTH (1-84). Specific binding of the hormone to the cells, which reached the equilibrium within 60 min, was observed. Half maximal binding was reached by 10 min. Binding was competitively inhibited by increasing doses of unlabeled PTH, and was about 55% displaced by adding, at the equilibrium, 10(-6) M unlabeled PTH. Autoradiography demonstrated specific label on the osteoclast. The cellular mechanism activated by the hormone remains to be elucidated.

  12. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  13. Tissue specificity of endothelin binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Bolger, G.T.; Liard, F.; Krogsrud, R.; Thibeault, D.; Jaramillo, J. (BioMega, Inc., Laval, Quebec (Canada))

    1990-09-01

    A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.

  14. New DNA-binding radioprotectors

    Science.gov (United States)

    Martin, Roger

    The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is

  15. The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence

    NARCIS (Netherlands)

    Holmes, AR; McNab, R; Millsap, KW; Rohde, M; Hammerschmidt, S; Mawdsley, JL; Jenkinson, HF

    Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins

  16. Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, O.M.; Wood, K.M.; Williams, D.C.

    1984-08-01

    Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

  17. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania

    2016-11-10

    Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlow\\'s drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

  18. Thermodynamics of Ligand Binding to Acyl-Coenzyme A Binding Protein Studied by Titration Calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils Joakim; Sigurskjold, Bent Walther; Kragelund, Birthe B.

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  19. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  20. Intentional binding of visual effects.

    Science.gov (United States)

    Ruess, Miriam; Thomaschke, Roland; Kiesel, Andrea

    2018-04-01

    When an action produces an effect, the effect is perceived earlier in time compared to a stimulus without preceding action. This temporal bias is called intentional binding (IB) and serves as an implicit measure of sense of agency. Typically, IB is investigated by presenting a rotating clock hand while participants execute an action and perceive a resulting tone. Participants are asked to estimate the time point of tone onset by referring to the clock hand position. This time point estimate is compared to a time point estimate of a tone in a condition in which the tone occurs without preceding action. Studies employing this classic clock paradigm employed auditory action effects. We modified this paradigm to investigate potential IB of visual action effects, and, additionally, to investigate how IB differs for visual action effects (Experiment 1) in comparison to auditory action effects (Experiment 2). Our results show that, like the IB of an auditory effect, the time point of a visual action effect is shifted toward the causing action, and that the size of the IB depends on the delay duration of the effect. Comparable to auditory action effects, earlier action effects showed stronger IB compared to later action effects. Yet overall IB of the visual effects was weaker than IB of the auditory effects. As IB is seen as an indicator of sense of agency, this may have important implications for the design of human-machine interfaces.

  1. Localization-enhanced biexciton binding in semiconductors

    DEFF Research Database (Denmark)

    Langbein, Wolfgang Werner; Hvam, Jørn Märcher

    1999-01-01

    The influence of excitonic localization on the binding energy of biexcitons is investigated for quasi-three-dimensional and quasi-two-dimensional AlxGa1-xAs structures. An increase of the biexciton binding energy is observed for localization energies comparable to or larger than the free biexcito...

  2. Neurotransmitter Receptor Binding in Bovine Cerebral Microvessels

    Science.gov (United States)

    Peroutka, Stephen J.; Moskowitz, Michael A.; Reinhard, John F.; Synder, Solomon H.

    1980-05-01

    Purified preparations of microvessels from bovine cerebral cortex contain substantial levels of alpha-adrenergic, beta-adrenergic, and histamine 1 receptor binding sites but only negligible serotonin, muscarinic cholinergic, opiate, and benzodiazepine receptor binding. Norepinephrine and histamine may be endogenous regulators of the cerebral microcirculation at the observed receptors.

  3. Identification of Glossina morsitans morsitans odorant binding ...

    African Journals Online (AJOL)

    Tsetse flies are vectors of trypanosome parasites, causative agents of Trypanosomiasis in humans and animals. Odorant Binding Proteins (OBPs) are critical in insect olfaction as they bind volatile odours from the environment and transport them to receptors within olfactory receptor neurons for processing providing critical ...

  4. Binding of corroded ions to human saliva.

    Science.gov (United States)

    Mueller, H J

    1985-05-01

    Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

  5. Factor VIIa binding and internalization in hepatocytes

    DEFF Research Database (Denmark)

    Hjortoe, G; Sorensen, B B; Petersen, L C

    2005-01-01

    The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2...... cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes...

  6. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  7. Ethylene binding site affinity in ripening apples

    Energy Technology Data Exchange (ETDEWEB)

    Blankenship, S.M. (North Carolina State Univ., Raleigh, NC (United States). Dept. of Horticultural Science); Sisler, E.C. (North Carolina State Univ., Raleigh, NC (United States))

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  8. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  9. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  10. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  11. Thermodynamic binding constants for gallium transferrin

    Energy Technology Data Exchange (ETDEWEB)

    Harris, W.R.; Pecoraro, V.L.

    1983-01-18

    Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well stablished that Ga/sup 3 +/ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO/sub 3/ have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO/sub 3/ are log K/sub 1/* = 20.3 and log K/sub 2/* = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe/sup 3 +/ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log (Ga(H/sub 2/O)/sub 6/)) to express the effective binding strength at pH 7.4.

  12. Myeloperoxidase selectively binds and selectively kills microbes.

    Science.gov (United States)

    Allen, Robert C; Stephens, Jackson T

    2011-01-01

    Myeloperoxidase (MPO) is reported to selectively bind to bacteria. The present study provides direct evidence of MPO binding selectivity and tests the relationship of selective binding to selective killing. The microbicidal effectiveness of H(2)O(2) and of OCl(-) was compared to that of MPO plus H(2)O(2). Synergistic microbicidal action was investigated by combining Streptococcus sanguinis, a H(2)O(2)-producing microbe showing low MPO binding, with high-MPO-binding Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa without exogenous H(2)O(2), with and without MPO, and with and without erythrocytes (red blood cells [RBCs]). Selectivity of MPO microbicidal action was conventionally measured as the MPO MIC and minimal bactericidal concentration (MBC) for 82 bacteria including E. coli, P. aeruginosa, S. aureus, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, and viridans streptococci. Both H(2)O(2) and OCl(-) destroyed RBCs at submicrobicidal concentrations. Nanomolar concentrations of MPO increased H(2)O(2) microbicidal action 1,000-fold. Streptococci plus MPO produced potent synergistic microbicidal action against all microbes tested, and RBCs caused only a small decrease in potency without erythrocyte damage. MPO directly killed H(2)O(2)-producing S. pyogenes but was ineffective against non-H(2)O(2)-producing E. faecalis. The MPO MICs and MBCs for E. coli, P. aeruginosa, and S. aureus were significantly lower than those for E. faecalis. The streptococcal studies showed much higher MIC/MBC results, but such testing required lysed horse blood-supplemented medium, thus preventing valid comparison of these results to those for the other microbes. E. faecalis MPO binding is reportedly weak compared to binding of E. coli, P. aeruginosa, and S. aureus but strong compared to binding of streptococci. Selective MPO binding results in selective killing.

  13. Druggability of methyl-lysine binding sites

    Science.gov (United States)

    Santiago, C.; Nguyen, K.; Schapira, M.

    2011-12-01

    Structural modules that specifically recognize—or read—methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions.

  14. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  15. Technetium-99m labeling and fibronectin binding ability of Corynebacterium diphtheriae; Marcacao de Corynebacterium diphtheriae com Tecnecio-99m e avaliacao da capacidade de ligacao a fibronectina de plasma humano

    Energy Technology Data Exchange (ETDEWEB)

    Souza, S.M.S.; Nagao, P.E.; Bernardo-Filho, M. [Universidade do Estado do Rio de Janeiro, RJ (Brazil). Inst. de Biologia Roberto Alcantara Gomes; Pereira, G.A.; Napoleao, F.; Andrade, A.F.B.; Hirata Junior, R.; Mattos-Guaraldi, A.L. [Universidade do Estado do Rio de Janeiro, RJ (Brazil). Faculdade de Ciencias Medicas

    2004-04-15

    The use of radionuclides has permitted advances in areas of clinical and scientific knowledge. Several molecules and cells have been labelled with Technetium-99m ({sup 99m}Tc). The stannous chloride (SnCl{sub 2}) has a significant influence on the labeling and stability of {sup 99m}Tc radiotracers. The frequent risk of diphtheria epidemics has intensified interest in the virulence factors of Corynebacterium diphtheriae. Although studies have looked at potential adhesins including haemagglutinins and exposed sugar residues, the molecular basis of mechanisms of adherence remains unclear. Adherence of pathogens to mammalian tissues may be mediated by fibronectin (FN) found in body fluids, matrix of connective tissues, and cell surfaces. In the present study we evaluated the binding ability to human plasma FN by {sup 99m}Tc labeled-C.diphtheriae. Due to adverse effects of stannous ions, microorganisms were submitted to survival and filamentation induction assays. Data showed a dose dependent susceptibility to SnCl{sub 2} bactericidal effects. Cell filamentation was observed for concentrations of SnCl{sub 2} > 110 {mu}g/ml. Adherence levels of {sup 99m}Tc labelled 241strain to coverslips coated with 20 {mu}g/ml FN were higher (P = 0.0037) than coated with bovine serum albumin. FN binding by the sucrose fermenting 241 C. diphtheriae strain (8.9% + 2.6) was significantly lower (P=0.0139) than Staphylococcus aureus Cowan I strain (34.1% {+-} 1.2). Therefore, bacterial {sup 99m}Tc labeling represents an additional tool that may contribute to the comprehension of C. diphtheriae interactions with host receptors such as FN that act as biological organizers by holding bacterial cells in position and guiding their migration. (author)

  16. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  17. HEAVY QUARK POTENTIALS AND QUARKONIA BINDING.

    Energy Technology Data Exchange (ETDEWEB)

    PETRECZKY,P.

    2004-11-04

    The author reviews recent progress in studying in-medium modification of inter-quark forces at finite temperature in lattice QCD. Some applications to the problem of quarkonium binding in potential models is also discussed.

  18. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  19. Adjustment of legally binding local plans

    DEFF Research Database (Denmark)

    Hvingel, Line Træholt; Aunsborg, Christian; Christensen, Finn Kjær

    2012-01-01

    Traditionally, and by law, new urban areas in Denmark are regulated and planned through legally binding local plans. Recently a tendency has occurred: The municipalities make the legally binding local plans quite open for future adjustment, and they are using a substantial amount of ‘empowerment......, which seem to be beyond the scope of the Danish Planning Act. This paper deals with this problem through case studies and a legal analysis of present law. If the combination of the legally binding local plan and subsequent added requirements is misused, it will weaken the legal rights of the citizens...... the considerations of legal rights, the extend of the legal use of empowerment provisions and the combination of the use of legal binding local plans and other legal instruments such as easements and sales agreements....

  20. Exciton Binding Energy of Monolayer WS2

    Science.gov (United States)

    Zhu, Bairen; Chen, Xi; Cui, Xiaodong

    2015-03-01

    The optical properties of monolayer transition metal dichalcogenides (TMDC) feature prominent excitonic natures. Here we report an experimental approach to measuring the exciton binding energy of monolayer WS2 with linear differential transmission spectroscopy and two-photon photoluminescence excitation spectroscopy (TP-PLE). TP-PLE measurements show the exciton binding energy of 0.71 +/- 0.01 eV around K valley in the Brillouin zone.

  1. Antimicrobial activities of heparin-binding peptides.

    OpenAIRE

    Andersson, Emma; Rydengård, Victoria; Sonesson, Andreas; Mörgelin, Matthias; Björck, Lars; Schmidtchen, Artur

    2004-01-01

    Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor...

  2. Electrostatically biased binding of kinesin to microtubules.

    Directory of Open Access Journals (Sweden)

    Barry J Grant

    2011-11-01

    Full Text Available The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.

  3. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  4. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  5. Identification of pseudomurein cell wall binding domains.

    Science.gov (United States)

    Steenbakkers, Peter J M; Geerts, Wim J; Ayman-Oz, Nilgün A; Keltjens, Jan T

    2006-12-01

    Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.

  6. The readiness potential reflects intentional binding

    Directory of Open Access Journals (Sweden)

    Han-Gue eJo

    2014-06-01

    Full Text Available When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP, which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with twenty mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  7. LIBRA: LIgand Binding site Recognition Application.

    Science.gov (United States)

    Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

    2015-12-15

    In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Zinc Binding by Lactic Acid Bacteria

    Directory of Open Access Journals (Sweden)

    Jasna Mrvčić

    2009-01-01

    Full Text Available Zinc is an essential trace element in all organisms. A common method for the prevention of zinc deficiency is pharmacological supplementation, especially in a highly available form of a metalloprotein complex. The potential of different microbes to bind essential and toxic heavy metals has recently been recognized. In this work, biosorption of zinc by lactic acid bacteria (LAB has been investigated. Specific LAB were assessed for their ability to bind zinc from a water solution. Significant amount of zinc ions was bound, and this binding was found to be LAB species-specific. Differences among the species in binding performance at a concentration range between 10–90 mg/L were evaluated with Langmuir model for biosorption. Binding of zinc was a fast process, strongly influenced by ionic strength, pH, biomass concentration, and temperature. The most effective metal-binding LAB species was Leuconostoc mesenteroides (27.10 mg of Zn2+ per gram of dry mass bound at pH=5 and 32 °C, during 24 h. FT-IR spectroscopy analysis and electron microscopy demonstrated that passive adsorption and active uptake of the zinc ions were involved.

  9. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  10. Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Nexo, E.

    1987-01-01

    Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L

  11. To Bind or not to Bind: It’s in the Contract

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    2015-01-01

    This article discusses the formalization of collaboration through partnering contracts in the construction industry in the USA, Great Britain and Denmark. The article compares the different types of collaborative partnering contracts in the three countries, and provides a conclusion on whether...... the collaborative partnering contract should be binding or non-binding, based on the three empirical contracts analyzed in this article. The partnering contracts in Great Britain and Denmark are legally binding, while in the USA the partnering agreements are non-binding charters or letters of intent. This article...... discusses, in a theoretical perspective, the legal reasoning behind the different partnering approaches, both from a historical and contract law perspective, and furthermore applies a game theoretical approach in evaluating binding versus non-binding partnering contracts. The analysis focuses on private...

  12. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  13. Are many Z-DNA binding proteins actually phospholipid-binding proteins?

    OpenAIRE

    Krishna, P; Kennedy, B P; Waisman, D M; van de Sande, J H; McGhee, J D

    1990-01-01

    We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In al...

  14. Structure-function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, Peter L.; Pedersen, Per Amstrup

    2000-01-01

    Na,K-ATPase; Mutagenesis; Na+ binding; K+ binding; Tl+ binding; Mg2+ binding; ATP binding; Cation binding site; Energy transduction......Na,K-ATPase; Mutagenesis; Na+ binding; K+ binding; Tl+ binding; Mg2+ binding; ATP binding; Cation binding site; Energy transduction...

  15. Mannose-binding geometry of pradimicin A.

    Science.gov (United States)

    Nakagawa, Yu; Doi, Takashi; Taketani, Takara; Takegoshi, K; Igarashi, Yasuhiro; Ito, Yukishige

    2013-08-05

    Pradimicins (PRMs) and benanomicins are the only family of non-peptidic natural products with lectin-like properties, that is, they recognize D-mannopyranoside (Man) in the presence of Ca(2+) ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti-HIV activities through binding to Man-containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate-forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM-A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM-A and Man. Evaluation of intermolecular distances by solid-state NMR spectroscopy revealed that the C2-C4 region of Man is in close contact with the primary binding site of PRM-A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co-precipitation experiments using deoxy-Man derivatives, leading to the proposal that PRM-A binds not only to terminal Man residues at the non-reducing end of glycans, but also to internal 6-substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM-A. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Alpine ski bindings and injuries. Current findings.

    Science.gov (United States)

    Natri, A; Beynnon, B D; Ettlinger, C F; Johnson, R J; Shealy, J E

    1999-07-01

    In spite of the fact that the overall incidence of alpine ski injuries has decreased during the last 25 years, the incidence of serious knee sprains usually involving the anterior cruciate ligament (ACL) has risen dramatically since the late 1970s. This trend runs counter to a dramatic reduction in lower leg injuries that began in the early 1970s and to date has lowered the risk of injury below the knee by almost 90%. One of the primary design objectives of modern ski boots and bindings has been to protect the skier from tibia and ankle fractures. So, in that sense, they have done an excellent job. However, despite advances in equipment design, modern ski bindings have not protected the knee from serious ligament trauma. At the present time, we are unaware of any binding design, settings or function that can protect both the knee and lower extremities from serious ligament sprains. No innovative change in binding design appears to be on the horizon that has the potential to reduce the risk of these severe knee injuries. Indeed, only 1 study has demonstrated a means to help reduce this risk of serious knee sprains, and this study involved education of skiers, not ski equipment. Despite the inability of bindings to reduce the risk of severe knee injuries there can be no doubt that improvement in ski bindings has been the most important factor in the marked reduction in incidence of lower leg and ankle injuries during the last 25 years. The authors strongly endorse the application of present International Standards Organisation (ISO) and American Society for Testing and Materials (ASTM) standards concerning mounting, setting and maintaining modern 'state of the art' bindings.

  17. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  18. The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

    Science.gov (United States)

    Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

    2007-02-23

    Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional

  19. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

    Full Text Available Low molecular weight chemical (LMW allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

  20. Computational search for aflatoxin binding proteins

    Science.gov (United States)

    Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

    2017-10-01

    Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

  1. Engineering Escherichia coli to bind to cyanobacteria.

    Science.gov (United States)

    Zhang, Zijian; Meng, Liuyi; Ni, Congjian; Yao, Lanqiu; Zhang, Fengyu; Jin, Yuji; Mu, Xuelang; Zhu, Shiyu; Lu, Xiaoyu; Liu, Shiyu; Yu, Congyu; Wang, Chenggong; Zheng, Pu; Wu, Jie; Kang, Li; Zhang, Haoqian M; Ouyang, Qi

    2017-03-01

    We engineered Escherichia coli cells to bind to cyanobacteria by heterologously producing and displaying lectins of the target cyanobacteria on their surface. To prove the efficacy of our approach, we tested this design on Microcystis aeruginosa with microvirin (Mvn), the lectin endogenously produced by this cyanobacterium. The coding sequence of Mvn was C-terminally fused to the ice nucleation protein NC (INPNC) gene and expressed in E. coli. Results showed that E. coli cells expressing the INPNC::Mvn fusion protein were able to bind to M. aeruginosa and the average number of E. coli cells bound to each cyanobacterial cell was enhanced 8-fold. Finally, a computational model was developed to simulate the binding reaction and help reconstruct the binding parameters. To our best knowledge, this is the first report on the binding of two organisms in liquid culture mediated by the surface display of lectins and it may serve as a novel approach to mediate microbial adhesion. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Assessment of the binding properties of granuloszint

    Energy Technology Data Exchange (ETDEWEB)

    Schubiger, P.A.; Hasler, P.H.; Novak-Hofer, I.; Blaeuenstein, P.

    1989-09-01

    /sup 123/I-granuloszint (a murine monoclonal antibody - called AK-47 - against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (nonspecific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of /sup 125/I-labelled AK-47 gave the following results: Affinity constant 5x10/sup 8/, 20,000-100,000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with /sup 123/I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro checks we have found a reliable way to control the production and to assure an optimal binding behaviour of /sup 123/I-granuloszint. (orig.).

  3. Human serum albumin binding of certain antimalarials

    Science.gov (United States)

    Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

    2018-03-01

    Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

  4. Binding of anandamide to bovine serum albumin

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2003-01-01

    The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a...... in aqueous compartments. - Bojesen, I. N., and H. S. Hansen. Binding of anandamide to bovine serum albumin.......The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because...... a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium...

  5. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  6. DNA binding studies of tartrazine food additive.

    Science.gov (United States)

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

    The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

  7. Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins.

    Science.gov (United States)

    Holth, L T; Sun, J M; Coutts, A S; Murphy, L C; Davie, J R

    1997-12-01

    The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.

  8. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-01-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

  9. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Salt-mediated two-site ligand binding by the cocaine-binding aptamer.

    Science.gov (United States)

    Neves, Miguel A D; Slavkovic, Sladjana; Churcher, Zachary R; Johnson, Philip E

    2017-02-17

    Multisite ligand binding by proteins is commonly utilized in the regulation of biological systems and exploited in a range of biochemical technologies. Aptamers, although widely utilized in many rationally designed biochemical systems, are rarely capable of multisite ligand binding. The cocaine-binding aptamer is often used for studying and developing sensor and aptamer-based technologies. Here, we use isothermal titration calorimetry (ITC) and NMR spectroscopy to demonstrate that the cocaine-binding aptamer switches from one-site to two-site ligand binding, dependent on NaCl concentration. The high-affinity site functions at all buffer conditions studied, the low-affinity site only at low NaCl concentrations. ITC experiments show the two ligand-binding sites operate independently of one another with different affinities and enthalpies. NMR spectroscopy shows the second binding site is located in stem 2 near the three-way junction. This ability to control ligand binding at the second site by adjusting the concentration of NaCl is rare among aptamers and may prove a useful in biotechnology applications. This work also demonstrates that in vitro selected biomolecules can have functions as complex as those found in nature. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

  12. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  13. Binding of alkylpyridinium chloride surfactants to sodium polystyrene sulfonate

    NARCIS (Netherlands)

    Ishiguro, M.; Koopal, L.K.

    2009-01-01

    Binding of cationic surfactants to anionic polymers is well studied. However, the surfactant binding characteristics at very low concentration near the start of binding and at high concentration, where charge compensation may Occur. are less well known. Therefore, the binding characteristics of

  14. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin

    2009-01-01

    The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin......, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment...... LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen...

  15. AUXIN BINDING PROTEIN1: the outsider.

    Science.gov (United States)

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-06-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence.

  16. The binding of Np to rat bone

    International Nuclear Information System (INIS)

    Ramounet, B.; Taylor, D.M.

    1997-01-01

    Neptunium has been shown to massively deposit in bone, after intravenous or intramuscular injections. Initially, it was uniformly distributed on periosteal and endosteal bone surfaces. The nature of the binding molecules, for this actinide, in the skeleton, has not yet been identified. The aim of this work was to characterize the ligands of neptunium by selective extractions of bone components. The preliminary results displayed the binding of 237 Np(IV) in the organic phase of bone, after intravenous or intramuscular contamination. Further studies are in progress, to quantify the fraction of Np bound to the organic and mineral compartment of bone, and to determine the affinity constant and the turn-over of the binding proteins. (authors)

  17. Mucin Binding Reduces Colistin Antimicrobial Activity

    Science.gov (United States)

    Huang, Johnny X.; Blaskovich, Mark A. T.; Pelingon, Ruby; Ramu, Soumya; Kavanagh, Angela; Elliott, Alysha G.; Butler, Mark S.

    2015-01-01

    Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance. PMID:26169405

  18. New binding mode to TNF-alpha revealed by ubiquitin-based artificial binding protein.

    Directory of Open Access Journals (Sweden)

    Andreas Hoffmann

    Full Text Available A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins--designed ankyrin repeat proteins--without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

  19. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  20. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been...... described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling...

  1. Binding energy of two-dimensional biexcitons

    DEFF Research Database (Denmark)

    Singh, Jai; Birkedal, Dan; Vadim, Lyssenko

    1996-01-01

    Using a model structure for a two-dimensional (2D) biexciton confined in a quantum well, it is shown that the form of the Hamiltonian of the 2D biexciton reduces into that of an exciton. The binding energies and Bohr radii of a 2D biexciton in its various internal energy states are derived...... analytically using the fractional dimension approach. The ratio of the binding energy of a 2D biexciton to that of a 2D exciton is found to be 0.228, which agrees very well with the recent experimental value. The results of our approach are compared with those of earlier theories....

  2. Binding energies of hypernuclei and hypernuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R. [Argonne National Lab., IL (United States)]|[Univ. of Illinois, Chicago, IL (United States). Dept. of Physics; Murali, S.; Usmani, Q.N. [Jamia Millia Islamia, New Delhi (India). Dept. of Physics

    1996-05-01

    In part 1 the effect of nuclear core dynamics on the binding energies of {Lambda} hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the {Lambda} single-particle energy in terms of basic {Lambda}-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body {Lambda}NN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei.

  3. Binding energies of hypernuclei and hypernuclear interactions

    International Nuclear Information System (INIS)

    Bodmer, A.R.; Univ. of Illinois, Chicago, IL; Murali, S.; Usmani, Q.N.

    1996-01-01

    In part 1 the effect of nuclear core dynamics on the binding energies of Λ hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the Λ single-particle energy in terms of basic Λ-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body ΛNN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei

  4. Myeloperoxidase Selectively Binds and Selectively Kills Microbes ▿ †

    OpenAIRE

    Allen, Robert C.; Stephens, Jackson T.

    2010-01-01

    Myeloperoxidase (MPO) is reported to selectively bind to bacteria. The present study provides direct evidence of MPO binding selectivity and tests the relationship of selective binding to selective killing. The microbicidal effectiveness of H2O2 and of OCl− was compared to that of MPO plus H2O2. Synergistic microbicidal action was investigated by combining Streptococcus sanguinis, a H2O2-producing microbe showing low MPO binding, with high-MPO-binding Escherichia coli, Staphylococcus aureus, ...

  5. Studies on folate binding and a radioassay for serum and whole blood folate using goat milk as binding agent

    International Nuclear Information System (INIS)

    Piyasena, R.D.; Weerasekera, D.A.; Hettiaratchi, N.; Wikramanayake, T.W.; Sri Lanka Univ., Peradeniya Campus. Nuclear Medicine Unit)

    1977-01-01

    Preparations of cow, goat, buffalo, and human milk in addition to pig plasma were tested for folate binding properties. Of these, only pig plasma and goat milk showed sufficient binding to enable use as binding agents in a radioassay for serum and whole blood folate. The binding of folate by cow mild preparations in particular was found to be very poor. (orig.) [de

  6. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  7. Binding sites analyser (BiSA: software for genomic binding sites archiving and overlap analysis.

    Directory of Open Access Journals (Sweden)

    Matloob Khushi

    Full Text Available Genome-wide mapping of transcription factor binding and histone modification reveals complex patterns of interactions. Identifying overlaps in binding patterns by different factors is a major objective of genomic studies, but existing methods to archive large numbers of datasets in a personalised database lack sophistication and utility. Therefore we have developed transcription factor DNA binding site analyser software (BiSA, for archiving of binding regions and easy identification of overlap with or proximity to other regions of interest. Analysis results can be restricted by chromosome or base pair overlap between regions or maximum distance between binding peaks. BiSA is capable of reporting overlapping regions that share common base pairs; regions that are nearby; regions that are not overlapping; and average region sizes. BiSA can identify genes located near binding regions of interest, genomic features near a gene or locus of interest and statistical significance of overlapping regions can also be reported. Overlapping results can be visualized as Venn diagrams. A major strength of BiSA is that it is supported by a comprehensive database of publicly available transcription factor binding sites and histone modifications, which can be directly compared to user data. The documentation and source code are available on http://bisa.sourceforge.net.

  8. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article d...

  10. Tension-induced binding of semiflexible biopolymers

    International Nuclear Information System (INIS)

    Benetatos, Panayotis; Heydt, Alice von der; Zippelius, Annette

    2014-01-01

    We investigate theoretically the effect of polymer tension on the collective behavior of reversibly binding cross-links. For this purpose, we employ a model of two weakly bending wormlike chains aligned in parallel by a tensile force, with a sequence of inter-chain binding sites regularly spaced along the contours. Reversible cross-links attach and detach at the sites with an affinity controlled by a chemical potential. In a mean-field approach, we calculate the free energy of the system and find the emergence of a free-energy barrier which controls the reversible (un)binding. The tension affects the conformational entropy of the chains which competes with the binding energy of the cross-links. This competition gives rise to a sudden increase in the fraction of bound sites as the tension increases. We show that this transition is related to the cross-over between weak and strong localization of a directed polymer in a pinning potential. The cross-over to the strongly bound state can be interpreted as a mechanism for force-stiffening which exceeds the capabilities of single-chain elasticity and thus available only to reversibly cross-linked polymers. (paper)

  11. Cross-Modal Binding in Developmental Dyslexia

    Science.gov (United States)

    Jones, Manon W.; Branigan, Holly P.; Parra, Mario A.; Logie, Robert H.

    2013-01-01

    The ability to learn visual-phonological associations is a unique predictor of word reading, and individuals with developmental dyslexia show impaired ability in learning these associations. In this study, we compared developmentally dyslexic and nondyslexic adults on their ability to form cross-modal associations (or "bindings") based…

  12. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

    The nucleoprotein structure of Arabidopsis thaliana telomeres was investigated. A protein specifically binding to telomeric sequences was characterized by gel mobility shift assays with synthetic oligonucleotides consisting of four 7 bp telomeric repeats of Arabidopsis (TTTAGGG) and crude nuclear protein extracts of Arabidopsis leaves. These DNA-protein binding studies revealed that the binding affinity of this telomere-binding protein to the G-rich single-strand as well as to the double-stranded telomeric DNA is much higher than to the C-rich single-strand. The molecular mass of the protein was identified by SDS-PAGE to be 67 kDa. The isoelectric points were determined to be 5.0, 4.85 and 4.7, respectively, indicating that either one protein with different modifications or three slightly different proteins have been isolated. An RNA component, possibly serving as a template for reverse transcription of a plant telomerase, does not mediate the DNA-protein contact because the DNA-protein interactions were not RNAse-sensitive.

  13. Binding of cationic surfactants to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Tan, W.; Koopal, L.K.

    2007-01-01

    Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the

  14. Ribosome binding site recognition using neural networks

    Directory of Open Access Journals (Sweden)

    Márcio Ferreira da Silva Oliveira

    2004-01-01

    Full Text Available Pattern recognition is an important process for gene localization in genomes. The ribosome binding sites are signals that can help in the identification of a gene. It is difficult to find these signals in the genome through conventional methods because they are highly degenerated. Artificial Neural Networks is the approach used in this work to address this problem.

  15. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    -density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low...

  16. Non-binding relationship between visual features

    Directory of Open Access Journals (Sweden)

    Dragan eRangelov

    2014-10-01

    Full Text Available The answer as to how visual attributes processed in different brain loci at different speeds are bound together to give us our unitary experience of the visual world remains unknown. In this study we investigated whether bound representations arise, as commonly assumed, through physiological interactions between cells in the visual areas. In a focal attentional task in which correct responses from either bound or unbound representations were possible, participants discriminated the colour or orientation of briefly presented single bars. On the assumption that representations of the two attributes are bound, the accuracy of reporting the colour and orientation should co-vary. By contrast, if the attributes are not mandatorily bound, the accuracy of reporting the two attributes should be independent. The results of our psychophysical studies reported here supported the latter, non-binding, relationship between visual features, suggesting that binding does not necessarily occur even under focal attention. We propose a task-contingent binding mechanism, postulating that binding occurs at late, post-perceptual, stages through the intervention of memory.

  17. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  18. The Double Bind: The next Generation

    Science.gov (United States)

    Malcom, Lindsey E.; Malcom, Shirley M.

    2011-01-01

    In this foreword, Shirley Malcom and Lindsey Malcom speak to the history and current status of women of color in science, technology, engineering, and mathematics (STEM) fields. As the author of the seminal report "The Double Bind: The Price of Being a Minority Woman in Science", Shirley Malcom is uniquely poised to give us an insightful…

  19. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

    Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (Ptannin (HT) level (Ptannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (Ptannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

  20. Alkali binding in hydrated Portland cement paste

    NARCIS (Netherlands)

    Chen, Wei; Brouwers, Jos

    2010-01-01

    The alkali-binding capacity of C–S–H in hydrated Portland cement pastes is addressed in this study. The amount of bound alkalis in C–S–H is computed based on the alkali partition theories firstly proposed by Taylor (1987) and later further developed by Brouwers and Van Eijk (2003). Experimental data

  1. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  2. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Binding site graphs: a new graph theoretical framework for prediction of transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Timothy E Reddy

    2007-05-01

    Full Text Available Computational prediction of nucleotide binding specificity for transcription factors remains a fundamental and largely unsolved problem. Determination of binding positions is a prerequisite for research in gene regulation, a major mechanism controlling phenotypic diversity. Furthermore, an accurate determination of binding specificities from high-throughput data sources is necessary to realize the full potential of systems biology. Unfortunately, recently performed independent evaluation showed that more than half the predictions from most widely used algorithms are false. We introduce a graph-theoretical framework to describe local sequence similarity as the pair-wise distances between nucleotides in promoter sequences, and hypothesize that densely connected subgraphs are indicative of transcription factor binding sites. Using a well-established sampling algorithm coupled with simple clustering and scoring schemes, we identify sets of closely related nucleotides and test those for known TF binding activity. Using an independent benchmark, we find our algorithm predicts yeast binding motifs considerably better than currently available techniques and without manual curation. Importantly, we reduce the number of false positive predictions in yeast to less than 30%. We also develop a framework to evaluate the statistical significance of our motif predictions. We show that our approach is robust to the choice of input promoters, and thus can be used in the context of predicting binding positions from noisy experimental data. We apply our method to identify binding sites using data from genome scale ChIP-chip experiments. Results from these experiments are publicly available at http://cagt10.bu.edu/BSG. The graphical framework developed here may be useful when combining predictions from numerous computational and experimental measures. Finally, we discuss how our algorithm can be used to improve the sensitivity of computational predictions of

  4. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  5. The Tomato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor I-2 Couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange

    NARCIS (Netherlands)

    Fenyk, S.; Dixon, C.H.; Gittens, W.H.; Townsend, P.D.; Sharples, G.J.; Pålsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognise and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception.

  6. Li+-ligand binding energies and the effect of ligand fluorination on the binding energies

    Science.gov (United States)

    Bauschlicher, Charles W.

    2018-02-01

    The Li+-ligand binding energies are computed for seven ligands and their perfluoro analogs using Density Functional Theory. The bonding is mostly electrostatic in origin. Thus the size of the binding energy tends to correlate with the ligand dipole moment, however, the charge-induced dipole contribution can be sufficiently large to affect the dipole-binding energy correlation. The perfluoro species are significantly less strongly bound than their parents, because the electron withdrawing power of the fluorine reduces the ligand dipole moment.

  7. Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

    Science.gov (United States)

    Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

    2017-04-04

    Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Rate Constants and Mechanisms of Protein-Ligand Binding.

    Science.gov (United States)

    Pang, Xiaodong; Zhou, Huan-Xiang

    2017-05-22

    Whereas protein-ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms.

  9. Cue integration and the perception of action in intentional binding

    DEFF Research Database (Denmark)

    Wolpe, Noham; Haggard, Patrick; Siebner, Hartwig R

    2013-01-01

    , further analyses showed that cue integration accounted for changes in action binding, but not tone binding. These findings establish a role for cue integration in action binding and support the growing evidence suggesting that action and tone binding are, at least in part, driven by distinct mechanisms....... that binding results from cue integration, in which a voluntary action provides information about the timing of its consequences or vice versa. The perception of the timing of either event is then a weighted average, determined according to the reliability of each of these two cues. Here we tested...... the contribution of cue integration to the perception of action and its sensory effect in binding, that is, action and tone binding, by manipulating the sensory reliability of the outcome tone. As predicted, when tone reliability was reduced, action binding was diminished and tone binding was increased. However...

  10. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  11. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  12. Is there a link between selectivity and binding thermodynamics profiles?

    Science.gov (United States)

    Tarcsay, Ákos; Keserű, György M

    2015-01-01

    Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...... nmol l-1 and 16 nmol l-1. The binding protein has a Stokes radius of 2.49 nm when saturated with cobalamin and 2.61 nm when unsaturated. It binds cobalamin over a broad range of pH and is able to bind cobinamide also. With immunohistochemistry, we find haptocorrin immunoreactivity in the mammary glands...

  14. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

  15. Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

    Science.gov (United States)

    Bach, Julia; Lewis, Nathaniel; Maggiora, Kathy; Gillespie, Alison J; Connell-Crowley, Lisa

    2015-08-28

    This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

    Science.gov (United States)

    Oh, Y S; Ahn, H S; Gye, M C

    2013-12-01

    Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse. © 2012 Blackwell Verlag GmbH.

  17. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  18. Causal binding of actions to their effects.

    Science.gov (United States)

    Buehner, Marc J; Humphreys, Gruffydd R

    2009-10-01

    According to widely held views in cognitive science harking back to David Hume, causality cannot be perceived directly, but instead is inferred from patterns of sensory experience, and the quality of these inferences is determined by perceivable quantities such as contingency and contiguity. We report results that suggest a reversal of Hume's conjecture: People's sense of time is warped by the experience of causality. In a stimulus-anticipation task, participants' response behavior reflected a shortened experience of time in the case of target stimuli participants themselves had generated, relative to equidistant, equally predictable stimuli they had not caused. These findings suggest that causality in the mind leads to temporal binding of cause and effect, and extend and generalize beyond earlier claims of intentional binding between action and outcome.

  19. Tight-binding treatment of conjugated polymers

    DEFF Research Database (Denmark)

    Lynge, Thomas Bastholm

    This PhD thesis concerns conjugated polymers which constitute a constantly growing research area. Today, among other things, conjugated polymers play a role in plastic based solar cells, photodetectors and light emitting diodes, and even today such plastic-based components constitute an alternative...... of tomorrow. This thesis specifically treats the three conjugated polymers trans-polyacetylene (tPA), poly(para-phenylene) (PPP) and poly(para-phe\\-nylene vinylene) (PPV). The present results, which are derived within the tight-binding model, are divided into two parts. In one part, analytic results...... are derived for the optical properties of the polymers expressed in terms of the optical susceptibility both in the presence and in the absence of a static electric field. In the other part, the cumputationally efficient Density Functional-based Tight-Binding (DFTB) model is applied to the description...

  20. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  1. Binding of 3H-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    International Nuclear Information System (INIS)

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.

    1985-01-01

    Binding of 3 H-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method

  2. Antimicrobial Peptides with Differential Bacterial Binding Characteristics

    Science.gov (United States)

    2013-03-01

    wherein each residue in the sequence is systematically replaced with alanine to produce a set of well-defined mutations, and 3) sequence generation...peptide sequences with differential binding behavior toward select microorganisms .  viii    Acknowledgements The authors would like to thank Mr. Steven...A., Sandri, L., & Giangaspero, A. (2000). Amphipathic, α-Helical Antimicrobial Peptides. Biopolymers , 55, 4-30.  2 Epand, R. M., & Vogel, H. J. (1999

  3. Iodide Binding in Sodium-Coupled Cotransporters.

    Science.gov (United States)

    Vergara-Jaque, Ariela; Fong, Peying; Comer, Jeffrey

    2017-12-26

    Several apical iodide translocation pathways have been proposed for iodide efflux out of thyroid follicular cells, including a pathway mediated by the sodium-coupled monocarboxylate transporter 1 (SMCT1), which remains controversial. Herein, we evaluate structural and functional similarities between SMCT1 and the well-studied sodium-iodide symporter (NIS) that mediates the first step of iodide entry into the thyroid. Free-energy calculations using a force field with electronic polarizability verify the presence of a conserved iodide-binding pocket between the TM2, TM3, and TM7 segments in hNIS, where iodide is coordinated by Phe67, Gln72, Cys91, and Gln94. We demonstrate the mutation of residue Gly93 of hNIS to a larger amino acid expels the side chain of a critical tryptophan residue (Trp255) into the interior of the binding pocket, partially occluding the iodide binding site and reducing iodide affinity, which is consistent with previous reports associating mutation of this residue with iodide uptake deficiency and hypothyroidism. Furthermore, we find that the position of Trp255 in this hNIS mutant mirrors that of Trp253 in wild-type hSMCT1, where a threonine (Thr91) occupies the position homologous to that occupied by glycine in wild-type hNIS (Gly93). Correspondingly, mutation of Thr91 to glycine in hSMCT1 makes the pocket structure more like that of wild-type hNIS, increasing its iodide affinity. These results suggest that wild-type hSMCT1 in the inward-facing conformation may bind iodide only very weakly, which may have implications for its ability to transport iodide.

  4. Asymmetric Aminalization via Cation-Binding Catalysis

    DEFF Research Database (Denmark)

    Park, Sang Yeon; Liu, Yidong; Oh, Joong Suk

    2018-01-01

    Asymmetric cation-binding catalysis, in principle, can generate "chiral" anionic nucleophiles, where the counter cations are coordinated within chiral environments. Nitrogen-nucleophiles are intrinsically basic, therefore, its use as nucleophiles is often challenging and limiting the scope...... of the reaction. Particularly, a formation of configurationally labile aminal centers with alkyl substituents has been a formidable challenge due to the enamine/imine equilibrium of electrophilic substrates. Herein, we report enantioselective nucleophilic addition reactions of potassium phthalimides to Boc-protected...

  5. Polypeptide binding properties of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C S; Heegaard, N H; Holm, A

    2000-01-01

    to be elucidated. We have investigated the interactions of human calreticulin with denatured ovalbumin, proteolytic digests of ovalbumin, and different available peptides by solid phase assays, size-exclusion chromatography, capillary electrophoresis, and MS. The results show that calreticulin interacts better...... with unfolded ovalbumin than with native ovalbumin, that calreticulin strongly binds components in proteolytic digests of denatured ovalbumin, and that calreticulin interacts strongly with certain synthetic peptides....

  6. What Happened to the IGF Binding Proteins?

    Science.gov (United States)

    Bach, Leon A

    2018-02-01

    Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

  7. Binding Energy and Equilibrium of Compact Objects

    Directory of Open Access Journals (Sweden)

    Germano M.

    2014-04-01

    Full Text Available The theoretical analysis of the existence of a limit mass for compact astronomic ob- jects requires the solution of the Einstein’s equations of g eneral relativity together with an appropriate equation of state. Analytical solutions exi st in some special cases like the spherically symmetric static object without energy sou rces that is here considered. Solutions, i.e. the spacetime metrics, can have a singular m athematical form (the so called Schwarzschild metric due to Hilbert or a nonsingula r form (original work of Schwarzschild. The former predicts a limit mass and, conse quently, the existence of black holes above this limit. Here it is shown that, the origi nal Schwarzschild met- ric permits compact objects, without mass limit, having rea sonable values for central density and pressure. The lack of a limit mass is also demonst rated analytically just imposing reasonable conditions on the energy-matter densi ty, of positivity and decreas- ing with radius. Finally the ratio between proper mass and to tal mass tends to 2 for high values of mass so that the binding energy reaches the lim it m (total mass seen by a distant observer. As it is known the negative binding energ y reduces the gravitational mass of the object; the limit of m for the binding energy provides a mechanism for stable equilibrium of any amount of mass to contrast the gravitatio nal collapse.

  8. The aesthetic experience of 'contour binding'.

    Science.gov (United States)

    Casco, Clara; Guzzon, Daniela

    2008-01-01

    To find the diagnostic spatial frequency information in different painting styles (cubism, impressionism and realism), we have compared sensitivity (d') in distinguishing signal (subject of the painting) from noise with normal, high-pass and low-pass filtered images at long (150 ms) and short (30 ms) exposure. We found that for cubist-style images, d' increases with high-pass filtering compared with normal and low-pass filtered images, but decreases with low-pass filtering compared with normal images. These results indicate that channels with high spatial resolution provide the diagnostic information to solve the binding problem. Sensitivity for images in impressionist style was instead reduced by both low- and high-pass filtering. This indicates that both high and low spatial frequency channels play a role in solving the binding problem, suggesting the involvement of large collator units that group the response of small channels tuned to the same orientation. The difference between realism, which shows higher sensitivity for low-frequency filtering at short durations and cubism in which the binding problem is solved by high spatial frequency channels, has a corresponding difference in aesthetic judgment: the probability of judging a painting as 'intriguing' is larger with low-pass filtering than with high-pass filtering in realism, while the opposite is true for cubism. This suggests that the aesthetic experience is available during early processing of an image, and could preferentially influence high-level categorization of the subject of a painting.

  9. Computation of pH-Dependent Binding Free Energies

    Science.gov (United States)

    Kim, M. Olivia; McCammon, J. Andrew

    2015-01-01

    Protein-ligand binding accompanies changes in the surrounding electrostatic environments of the two binding partners and may lead to changes in protonation upon binding. In cases where the complex formation results in a net transfer of protons, the binding process is pH-dependent. However, conventional free energy computations or molecular docking protocols typically employ fixed protonation states for the titratable groups in both binding partners set a priori, which are identical for the free and bound states. In this review, we draw attention to these important yet largely ignored binding-induced protonation changes in protein-ligand association by outlining physical origins and prevalence of the protonation changes upon binding. Following a summary of various theoretical methods for pKa prediction, we discuss the theoretical framework to examine the pH dependence of protein-ligand binding processes. PMID:26202905

  10. Genetics Home Reference: core binding factor acute myeloid leukemia

    Science.gov (United States)

    ... acute myeloid leukemia Core binding factor acute myeloid leukemia Printable PDF Open All Close All Enable Javascript ... collapse boxes. Description Core binding factor acute myeloid leukemia (CBF-AML) is one form of a cancer ...

  11. Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

    Science.gov (United States)

    Yu, M; Qi, R; Chen, C; Yin, J; Ma, S; Shi, W; Wu, Y; Ge, J; Jiang, Y; Tang, L; Xu, Y; Li, Y

    2017-02-01

    The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants. Genetically engineered Lactobacillus casei strains expressing F4 (K88) fimbrial adhesin FaeG (rLpPG-2-FaeG) and either co-expressing heat-labile enterotoxin A (LTA) subunit with an amino acid mutation associated with reduced virulence (LTAK63) and a heat-labile enterotoxin B (LTB) subunit of E. coli (rLpPG-2-LTAK63-co-LTB) or fused-expressing LTAK63 and LTB (rLpPG-2-LTAK63-fu-LTB) were constructed. The immunogenicity of rLpPG-2-FaeG in conjunction with rLpPG-2-LTAK63-co-LTB or rLpPG-2-LTAK63-fu-LTB as an orally administered mucosal adjuvant in mice was evaluated. Results showed that the levels of FaeG-specific serum IgG and mucosal sIgA, as well as the proliferation of lymphocytes, were significantly higher in mice orally co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB compared with those administered rLpPG-2-FaeG alone, and were lower than those co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB. Moreover, effective protection was observed after challenge with F4+ ETEC strain CVCC 230 in mice co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB or rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB group compared with those that received rLpPG-2-FaeG alone. rLpPG-2-FaeG showed greater immunogenicity in combination with LTAK63 and LTB as molecular adjuvants. Recombinant Lactobacillus provides a promising platform for the development of vaccines against F4+ ETEC infection. © 2016 The Society for Applied Microbiology.

  12. Binding Studies of Lamotrigine with Sera of Different Animal Species

    African Journals Online (AJOL)

    Erah

    into the mechanism of interaction, evaluate the effect of dielectric constant on binding affinity, and to determine the effect of ..... Physico-chemical aspects of protein binding of nimesulide, Ind J. Pharm Sci, 2005; 2: 243-246. 10. Dutta, SK, Basu, SK, Sen KK. Binding of diclofenac sodium with bovine serum albumin at different ...

  13. Atomistic fingerprint of hyaluronan-CD44 binding

    DEFF Research Database (Denmark)

    Vuorio, Joni; Vattulainen, Ilpo; Martinez-Seara, Hector

    2017-01-01

    that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding...

  14. Binding of Intrinsic and Extrinsic Features in Working Memory

    Science.gov (United States)

    Ecker, Ullrich K. H.; Maybery, Murray; Zimmer, Hubert D.

    2013-01-01

    There is ongoing debate concerning the mechanisms of feature binding in working memory. In particular, there is controversy regarding the extent to which these binding processes are automatic. The present article demonstrates that binding mechanisms differ depending on whether the to-be-integrated features are perceived as forming a coherent…

  15. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  16. A sequential binding mechanism in a PDZ domain

    DEFF Research Database (Denmark)

    Chi, Celestine N; Bach, Anders; Engström, Åke

    2009-01-01

    that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants...

  17. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  18. NMR studies on DNA binding specificity of the lac repressor

    NARCIS (Netherlands)

    Kopke Salinas, Roberto

    2005-01-01

    The thesis describes NMR structures of two protein-DNA complexes. The first structure shows how the protein, the DNA binding domain of lac repressor, recognizes its natural DNA binding site, by adaptation and read out of the nucleotide sequence. The second one shows how the DNA binding specificity

  19. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic acids...

  20. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. (Lawrence Berkeley Lab., CA (United States))

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  1. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  2. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

  3. Zooming into the binding groove of HLA molecules : which positions and which substitutions change peptide binding most?

    NARCIS (Netherlands)

    van Deutekom, Hanneke W M; Kesmir, C.

    Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome. Almost all polymorphic residues are located in the peptide-binding groove, resulting in different peptide-binding preferences. Whether a single amino acid change can alter the peptide-binding repertoire of an HLA

  4. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B

    1999-01-01

    spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...... is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration...

  5. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    modulator of the GABAA receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we......Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous....... There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  6. Autoradiographic localization of benzomorphan binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Crain, B.J.; Kwenjen Chang; McNamara, J.O.; Valdes, F.

    1985-07-17

    The benzomorphan subpopulation of opiate binding sites was labeled by (TH)diprenorphine in the presence of unlabeled ligands selected to quench and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. The distribution differs from the distributions of , delta and kappa opiate binding and is quite similar to the distribution of US -endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. (Auth.). 34 refs.; 3 figs.

  7. Dual chain synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  8. Helical propensity in an intrinsically disordered protein accelerates ligand binding

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Dogan, Jakob; Jemth, Per

    2014-01-01

    domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure......Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation...... the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins....

  9. Structural basis for PECAM-1 homophilic binding

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, Cathy; Zhou, Dongwen; Lertkiatmongkol, Panida; Newman, Peter J.; Zhu, Jieqing (MCW)

    2015-12-23

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1–mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1–mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1–PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a β-sandwich topology formed by 2 sheets of antiparallel β strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å2. These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.

  10. Antioxidant flavonoids bind human serum albumin

    Science.gov (United States)

    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  11. End binding proteins are obligatory dimers.

    Directory of Open Access Journals (Sweden)

    Indrani Sen

    Full Text Available End binding (EB proteins are responsible for the recruitment of an array of microtubule plus-end tracking proteins (+TIPs to growing microtubules ends. EBs encompass an N-terminal calponin homology domain that confers microtubule tip tracking activity to the protein. The C-terminal domain of EBs contains a coiled coil that mediates the parallel dimerization of EB monomers. This part of the protein is also responsible for partner binding. While dimerization is not essential for microtubule tip tracking by EBs it is a prerequisite for +TIP partner binding. The concentration of EBs in cells has been estimated to be in the range of hundreds of nanomoles. In contrast, in in vitro single molecule experiments EB concentrations of subnanomoles are employed. From a mechanistic point of view it is important to assess the oligomerization state of EBs at physiologically and experimentally relevant protein concentrations, in particular if the goal of a study is to model the behavior of EB-dependent dynamic +TIP networks. Here we have determined the stability of the EB1 and EB3 dimers using multi-angle light scattering and fluorescence analytical ultracentrifugation. We show that these EBs form stable dimers and do not dissociate even at very low nanomolar concentrations. The dimers remained stable at both room temperature as well as at the physiologically relevant temperature of 37°C. Together, our results reveal that EBs are obligatory dimers, a conclusion that has implications for the mechanistic understanding of these key proteins involved in the orchestration of dynamic protein networks at growing microtubule ends.

  12. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  13. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  14. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  15. Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

    International Nuclear Information System (INIS)

    Young, R.A.; Meyers, B.; Alex, S.; Fang, S.L.; Braverman, L.E.

    1988-01-01

    The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

  16. An Integrated Computational Approach to Binding Theory

    OpenAIRE

    Bonato, Roberto

    2006-01-01

    thèse en cotutelle franco italienne; bourse régionale de la Vénétie; Pronouns play a decisive role in every natural language as the linguistic ele- ments that enable semantic cohesion of a text. Anaphora resolution (that is, the task of recovering in an automatic way the semantic content of pronouns) is therefore both an important theoretical issue and a ma jor technological challenge for any computer application that aims at a ?ner-grained semantic analysis of natural language texts. Binding...

  17. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch...... is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

  18. Triazatriangulene as binding group for molecular electronics

    DEFF Research Database (Denmark)

    Wei, Zhongming; Wang, Xintai; Borges, Anders

    2014-01-01

    The triazatriangulene (TATA) ring system was investigated as a binding group for tunnel junctions of molecular wires on gold surfaces. Self-assembled monolayers (SAMs) of TATA platforms with three different lengths of phenylene wires were fabricated, and their electrical conductance was recorded...... platform displays a contact resistance only slightly larger than the thiols. This surprising finding has not been reported before and was analyzed by theoretical computations of the transmission functions of the TATA anchored molecular wires. The relatively low contact resistance of the TATA platform along...

  19. DNS and BIND on IPv6

    CERN Document Server

    Liu, Cricket

    2011-01-01

    If you're preparing to roll out IPv6 on your network, this concise book provides the essentials you need to support this protocol with DNS. You'll learn how DNS was extended to accommodate IPv6 addresses, and how you can configure a BIND name server to run on the network. This book also features methods for troubleshooting problems with IPv6 forward- and reverse-mapping, and techniques for helping islands of IPv6 clients communicate with IPv4 resources. Topics include: DNS and IPv6-Learn the structure and representation of IPv6 addresses, and the syntaxes of AAAA and PTR records in the ip6.a

  20. RNA Helicases at work: binding and rearranging

    Science.gov (United States)

    Jankowsky, Eckhard

    2010-01-01

    RNA helicases are ubiquitous, highly conserved enzymes that participate in nearly all aspects of RNA metabolism. These proteins bind or remodel RNA or RNA–protein complexes in an ATP-dependent fashion. How RNA helicases physically perform their cellular tasks has been a longstanding question, but in recent years, intriguing models have started to link structure, mechanism and biological function for some RNA helicases. This review outlines our current view on major structural and mechanistic themes of RNA helicase function, and on emerging physical models for cellular roles of these enzymes. PMID:20813532

  1. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  2. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  3. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

  4. Binding of diphtheria toxin to phospholipids in liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-01-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine/cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  5. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  6. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  7. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Generowicz, Jacek; Lavrijsen, Wim T.L.P.; Marino, Massimo; Mato, Pere

    2004-01-01

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

  9. Maximizing binding capacity for protein A chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Zhang, Jennifer; Conley, Lynn; Caple, Ryan; Williams, Kevin P; Cecchini, Douglas

    2014-01-01

    Advances in cell culture expression levels in the last two decades have resulted in monoclonal antibody titers of ≥10 g/L to be purified downstream. A high capacity capture step is crucial to prevent purification from being the bottleneck in the manufacturing process. Despite its high cost and other disadvantages, Protein A chromatography still remains the optimal choice for antibody capture due to the excellent selectivity provided by this step. A dual flow loading strategy was used in conjunction with a new generation high capacity Protein A resin to maximize binding capacity without significantly increasing processing time. Optimum conditions were established using a simple empirical Design of Experiment (DOE) based model and verified with a wide panel of antibodies. Dynamic binding capacities of >65 g/L could be achieved under these new conditions, significantly higher by more than one and half times the values that have been typically achieved with Protein A in the past. Furthermore, comparable process performance and product quality was demonstrated for the Protein A step at the increased loading. © 2014 American Institute of Chemical Engineers.

  10. Microtubule binding distinguishes dystrophin from utrophin

    Science.gov (United States)

    Belanto, Joseph J.; Mader, Tara L.; Eckhoff, Michael D.; Strandjord, Dana M.; Banks, Glen B.; Gardner, Melissa K.; Lowe, Dawn A.; Ervasti, James M.

    2014-01-01

    Dystrophin and utrophin are highly similar proteins that both link cortical actin filaments with a complex of sarcolemmal glycoproteins, yet localize to different subcellular domains within normal muscle cells. In mdx mice and Duchenne muscular dystrophy patients, dystrophin is lacking and utrophin is consequently up-regulated and redistributed to locations normally occupied by dystrophin. Transgenic overexpression of utrophin has been shown to significantly improve aspects of the disease phenotype in the mdx mouse; therefore, utrophin up-regulation is under intense investigation as a potential therapy for Duchenne muscular dystrophy. Here we biochemically compared the previously documented microtubule binding activity of dystrophin with utrophin and analyzed several transgenic mouse models to identify phenotypes of the mdx mouse that remain despite transgenic utrophin overexpression. Our in vitro analyses revealed that dystrophin binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin has no activity in either assay. We also found that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, loss of torque production after in vivo eccentric contractions, or physical inactivity after mild exercise. Finally, our data suggest that exercise-induced inactivity correlates with loss of sarcolemmal neuronal NOS localization in mdx muscle, whereas loss of in vivo torque production after eccentric contraction-induced injury is associated with microtubule lattice disorganization. PMID:24706788

  11. Ice-Binding Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Melissa Bredow

    2017-12-01

    Full Text Available Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs, that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops.

  12. Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate

    International Nuclear Information System (INIS)

    Marks, M.J.; Collins, A.C.

    1982-01-01

    The binding of [ 3 H]nicotine to mouse brain has been measured and subsequently compared with the binding of [ 125 I]alpha-bungarotoxin (alpha-BTX) and L-[ 3 H]quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. The average KD and Bmax were 59 nM and 88 fmoles/mg of protein, respectively. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. When measured at 20 degrees or 37 degrees, nicotine appeared to bind to a single class of binding sites, but a second, very low-affinity, binding site was observed at 4 degrees. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl 2 , or MgSO 4 to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrain and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors

  13. The role of adhesins in bacteria motility modification

    Science.gov (United States)

    Conrad, Jacinta; Gibiansky, Maxsim; Jin, Fan; Gordon, Vernita; Motto, Dominick; Shrout, Joshua; Parsek, Matthew; Wong, Gerard

    2010-03-01

    Bacterial biofilms are multicellular communities responsible for a broad range of infections. To investigate the early-stage formation of biofilms, we have developed high-throughput techniques to quantify the motility of surface-associated bacteria. We translate microscopy movies of bacteria into a searchable database of trajectories using tracking algorithms adapted from colloidal physics. By analyzing the motion of both wild-type (WT) and isogenic knockout mutants, we have previously characterized fundamental motility mechanisms in P. aeruginosa. Here, we develop biometric routines to recognize signatures of adhesion and trapping. We find that newly attached bacteria move faster than previously adherent bacteria, and are more likely to be oriented out-of-plane. Motility appendages influence the bacterium's ability to become trapped: WT bacteria exhibit two types of trapped trajectories, whereas flagella-deficient bacteria rarely become trapped. These results suggest that flagella play a key role in adhesion.

  14. Curli fimbria: an Escherichia coli adhesin associated with human cystitis.

    Science.gov (United States)

    Cordeiro, Melina Aparecida; Werle, Catierine Hirsch; Milanez, Guilherme Paier; Yano, Tomomasa

    2016-01-01

    Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-d-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  15. Adhesins of immunoglobulin-like superfamily from earthworm Eisenia foetida.

    Science.gov (United States)

    Popović, M; Hrzenjak, T; Grdisa, M; Vuković, S

    1998-05-01

    1. From the biologically active extract (G-90) isolated from the tissue homogenate of Eisenia foetida immunoglobulin-like structures were isolated and named G-90/4. 2. G-90/4 in nanogram concentrations stimulated cell proliferation more than did the original G-90. It lyses cells in microgram concentrations. 3. G-90/4 acts as an adhesion molecule between the receptors of adjacent cells. 4. The increase in proliferative activity was accompanied by the elevation of cytoplasmic protein containing tyrosine. 5. Immunohistochemical analyses confirm immunoglobulin-like transmembrane structures in the connective and muscular tissues of E. foetida.

  16. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions...

  17. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  18. Thermodynamics of nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong ADP binding.

    Science.gov (United States)

    Robblee, James P; Cao, Wenxiang; Henn, Arnon; Hannemann, Diane E; De La Cruz, Enrique M

    2005-08-02

    We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.

  19. Implications of melanin binding in ocular drug delivery.

    Science.gov (United States)

    Rimpelä, Anna-Kaisa; Reinisalo, Mika; Hellinen, Laura; Grazhdankin, Evgeni; Kidron, Heidi; Urtti, Arto; Del Amo, Eva M

    2017-12-13

    Pigmented ocular tissues contain melanin within the intracellular melanosomes. Drugs bind to melanin at varying extent that ranges from no binding to extensive binding. Binding may lead to drug accumulation to the pigmented tissues and prolonged drug retention in the melanin containing cells. Therefore, melanin binding is an important feature that affects ocular drug delivery and biodistribution, but this topic has not been reviewed since 1998. In this review, we present current knowledge on ocular melanin, melanosomes and binding of drugs to pigmented cells and tissues. In vitro, in vivo and in silico methods in the field were critically evaluated, because the literature in this field can be confusing if the reader does not properly understand the methodological aspects. Literature analysis includes a comprehensive table of literature data on melanin binding of drugs. Furthermore, we aimed to give some insights beyond the current literature by making a chemical structure based classification model for melanin binding of drugs and kinetic simulations that revealed significant interplay between melanin binding and drug permeability across the melanosomal and plasma membranes. Overall, more mechanistic and systematic research is needed before the impact of melanin binding on ocular drug delivery can be properly understood and predicted. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  1. An Electrostatic Funnel in the GABA-Binding Pathway.

    Directory of Open Access Journals (Sweden)

    Timothy S Carpenter

    2016-04-01

    Full Text Available The γ-aminobutyric acid type A receptor (GABAA-R is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a 'funnel' that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site.

  2. Binding biological motion and visual features in working memory.

    Science.gov (United States)

    Ding, Xiaowei; Zhao, Yangfan; Wu, Fan; Lu, Xiqian; Gao, Zaifeng; Shen, Mowei

    2015-06-01

    Working memory mechanisms for binding have been examined extensively in the last decade, yet few studies have explored bindings relating to human biological motion (BM). Human BM is the most salient and biologically significant kinetic information encountered in everyday life and is stored independently from other visual features (e.g., colors). The current study explored 3 critical issues of BM-related binding in working memory: (a) how many BM binding units can be retained in working memory, (b) whether involuntarily object-based binding occurs during BM binding, and (c) whether the maintenance of BM bindings in working memory requires attention above and beyond that needed to maintain the constituent dimensions. We isolated motion signals of human BM from non-BM sources by using point-light displays as to-be-memorized BM and presented the participants colored BM in a change detection task. We found that working memory capacity for BM-color bindings is rather low; only 1 or 2 BM-color bindings could be retained in working memory regardless of the presentation manners (Experiments 1-3). Furthermore, no object-based encoding took place for colored BM stimuli regardless of the processed dimensions (Experiments 4 and 5). Central executive attention contributes to the maintenance of BM-color bindings, yet maintaining BM bindings in working memory did not require more central attention than did maintaining the constituent dimensions in working memory (Experiment 6). Overall, these results suggest that keeping BM bindings in working memory is a fairly resource-demanding process, yet central executive attention does not play a special role in this cross-module binding. (c) 2015 APA, all rights reserved).

  3. Apolipoprotein B is a calcium binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Dashti, N.; Lee, D.M.; Mok, T.

    1986-05-29

    Human hepatocarcinoma Hep G2 cells were grown in culture medium containing (/sup 45/Ca/sup 2 +/). The secreted lipoproteins of d < 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of (/sup 45/Ca) from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated (/sup 45/Ca/sup 2 +/) and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.

  4. A Cationic Smart Copolymer for DNA Binding

    Directory of Open Access Journals (Sweden)

    Tânia Ribeiro

    2017-11-01

    Full Text Available A new block copolymer with a temperature-responsive block and a cationic block was prepared by reversible addition-fragmentation chain transfer (RAFT polymerization, with good control of its size and composition. The first block is composed by di(ethylene glycol methyl ether methacrylate (DEGMA and oligo(ethylene glycol methyl ether methacrylate (OEGMA, with the ratio DEGMA/OEGMA being used to choose the volume phase transition temperature of the polymer in water, tunable from ca. 25 to above 90 °C. The second block, of trimethyl-2-methacroyloxyethylammonium chloride (TMEC, is positively charged at physiological pH values and is used for DNA binding. The coacervate complexes between the block copolymer and a model single strand DNA are characterized by fluorescence correlation spectroscopy and fluorescence spectroscopy. The new materials offer good prospects for biomedical application, for example in controlled gene delivery.

  5. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  6. Binding of radionuclides to proteins in fish

    International Nuclear Information System (INIS)

    Suzuki, Yuzuru

    1981-01-01

    Radioisotope tracer experiments on binding of radionuclides to proteins in fish were carried out in order to gain further information on biochemical behavior of radionuclides in marine fish. The radionuclides, which were bound to proteins in fish through sea water and food, were extracted with a Trisacetate buffer solution and separated by gel filtration on Sephadex G-75. Most of 137 Cs in the fish liver were bound only to a peptide with a molecular weight of 1,100 - 1,300. The most remarkable feature of 60 Co in the profiles of the gel filtration was the presence of two clear radioactivity pearks and the radioactivity appeared to transfer from a low molecular weight protein to a high molecular weight protein in the case of the uptake, and the reverse phenomenon was observed in the case of the excretion. Therefore, this suggested that these proteins had each inherent turnover rate for 60 Co. The profiles of the gel filtration of 65 Zn varied widely among species of fish, tissues or organs even in the same fish and pathways of the uptake. 125 I was bound to a relatively low molecular weight substance in cultured eel, however, the binding of 125 I to a protein with higher molecular weight was observed in the eel head including thyroid gland marked through food, and this protein was estimated to be thyroglobulin with molecular weight of 670,000. Although 95 Nb, 144 Ce- 144 Pr and 106 Ru- 106 Rh probably have no biological function in fish, it was apparently found to be organically bound in tissues or organs of the marine fish. (author)

  7. Heavy metals binding properties of esterified lemon

    International Nuclear Information System (INIS)

    Arslanoglu, Hasan; Altundogan, Hamdi Soner; Tumen, Fikret

    2009-01-01

    Sorption of Cd 2+ , Cr 3+ , Cu 2+ , Ni 2+ , Pb 2+ and Zn 2+ onto a carboxyl groups-rich material prepared from lemon was investigated in batch systems. The results revealed that the sorption is highly pH dependent. Sorption kinetic data indicated that the equilibrium was achieved in the range of 30-240 min for different metal ions and sorption kinetics followed the pseudo-second-order model for all metals studied. Relative sorption rate of various metal cations was found to be in the general order of Ni 2+ > Cd 2+ > Cu 2+ > Pb 2+ > Zn 2+ > Cr 3+ . The binding characteristics of the sorbent for heavy metal ions were analyzed under various conditions and isotherm data was accurately fitted to the Langmuir equation. The metal binding capacity order calculated from Langmuir isotherm was Pb 2+ > Cu 2+ > Ni 2+ > Cd 2+ > Zn 2+ > Cr 3+ . The mean free energy of metal sorption process calculated from Dubinin-Radushkevich parameter and the Polanyi potential was found to be in the range of 8-11 kJ mol -1 for the metals studied showing that the main mechanism governing the sorption process seems to be ion exchange. The basic thermodynamic parameters of metals ion sorption process were calculated by using the Langmuir constants obtained from equilibration study. The ΔG o and ΔH o values for metals ion sorption on the lemon sorbent showed the process to be spontaneous and exothermic in nature. Relatively low ΔH o values revealed that physical adsorption significantly contributed to the mechanism.

  8. Heavy metals binding properties of esterified lemon.

    Science.gov (United States)

    Arslanoglu, Hasan; Altundogan, Hamdi Soner; Tumen, Fikret

    2009-05-30

    Sorption of Cd(2+), Cr(3+), Cu(2+), Ni(2+), Pb(2+) and Zn(2+) onto a carboxyl groups-rich material prepared from lemon was investigated in batch systems. The results revealed that the sorption is highly pH dependent. Sorption kinetic data indicated that the equilibrium was achieved in the range of 30-240 min for different metal ions and sorption kinetics followed the pseudo-second-order model for all metals studied. Relative sorption rate of various metal cations was found to be in the general order of Ni(2+)>Cd(2+)>Cu(2+)>Pb(2+)>Zn(2+)>Cr(3+). The binding characteristics of the sorbent for heavy metal ions were analyzed under various conditions and isotherm data was accurately fitted to the Langmuir equation. The metal binding capacity order calculated from Langmuir isotherm was Pb(2+)>Cu(2+)>Ni(2+)>Cd(2+)>Zn(2+)>Cr(3+). The mean free energy of metal sorption process calculated from Dubinin-Radushkevich parameter and the Polanyi potential was found to be in the range of 8-11 kJ mol(-1) for the metals studied showing that the main mechanism governing the sorption process seems to be ion exchange. The basic thermodynamic parameters of metals ion sorption process were calculated by using the Langmuir constants obtained from equilibration study. The DeltaG degrees and DeltaH degrees values for metals ion sorption on the lemon sorbent showed the process to be spontaneous and exothermic in nature. Relatively low DeltaH degrees values revealed that physical adsorption significantly contributed to the mechanism.

  9. Heavy metals binding properties of esterified lemon

    Energy Technology Data Exchange (ETDEWEB)

    Arslanoglu, Hasan; Altundogan, Hamdi Soner [Department of Chemical Engineering, Firat University, 23279 Elazig (Turkey); Tumen, Fikret, E-mail: ftumen@firat.edu.tr [Department of Chemical Engineering, Firat University, 23279 Elazig (Turkey)

    2009-05-30

    Sorption of Cd{sup 2+}, Cr{sup 3+}, Cu{sup 2+}, Ni{sup 2+}, Pb{sup 2+} and Zn{sup 2+} onto a carboxyl groups-rich material prepared from lemon was investigated in batch systems. The results revealed that the sorption is highly pH dependent. Sorption kinetic data indicated that the equilibrium was achieved in the range of 30-240 min for different metal ions and sorption kinetics followed the pseudo-second-order model for all metals studied. Relative sorption rate of various metal cations was found to be in the general order of Ni{sup 2+} > Cd{sup 2+} > Cu{sup 2+} > Pb{sup 2+} > Zn{sup 2+} > Cr{sup 3+}. The binding characteristics of the sorbent for heavy metal ions were analyzed under various conditions and isotherm data was accurately fitted to the Langmuir equation. The metal binding capacity order calculated from Langmuir isotherm was Pb{sup 2+} > Cu{sup 2+} > Ni{sup 2+} > Cd{sup 2+} > Zn{sup 2+} > Cr{sup 3+}. The mean free energy of metal sorption process calculated from Dubinin-Radushkevich parameter and the Polanyi potential was found to be in the range of 8-11 kJ mol{sup -1} for the metals studied showing that the main mechanism governing the sorption process seems to be ion exchange. The basic thermodynamic parameters of metals ion sorption process were calculated by using the Langmuir constants obtained from equilibration study. The {Delta}G{sup o} and {Delta}H{sup o} values for metals ion sorption on the lemon sorbent showed the process to be spontaneous and exothermic in nature. Relatively low {Delta}H{sup o} values revealed that physical adsorption significantly contributed to the mechanism.

  10. Specific binding of beta-endorphin to normal human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  11. Hardware device to physical structure binding and authentication

    Science.gov (United States)

    Hamlet, Jason R.; Stein, David J.; Bauer, Todd M.

    2013-08-20

    Detection and deterrence of device tampering and subversion may be achieved by including a cryptographic fingerprint unit within a hardware device for authenticating a binding of the hardware device and a physical structure. The cryptographic fingerprint unit includes an internal physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generate an internal PUF value. Binding logic is coupled to receive the internal PUF value, as well as an external PUF value associated with the physical structure, and generates a binding PUF value, which represents the binding of the hardware device and the physical structure. The cryptographic fingerprint unit also includes a cryptographic unit that uses the binding PUF value to allow a challenger to authenticate the binding.

  12. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  13. Binding Characteristics Of Ivermectin To Blood Cells | Nweke ...

    African Journals Online (AJOL)

    The binding characteristics of Ivermectin were determined using scatchard plots. The percentage binding to platelet rich plasma, white blood cells and red blood cells were 90.00 + 1.00, 96-90 + 1.05 and 46.20 + 1.10 S.D respectively. It was found to bind the highest to white blood cells and the least to red blood cells.

  14. OCTAMER-BINDING TRANSCRIPTION FACTORS: GENOMICS AND FUNCTIONS

    OpenAIRE

    Zhao, Feng-Qi

    2013-01-01

    The Octamer-binding proteins (Oct) are a group of highly conserved transcription factors that specifically bind to the octamer motif (ATGCAAAT) and closely related sequences that are found in promoters and enhancers of a wide variety of both ubiquitously expressed and cell type-specific genes. Oct factors belong to the larger family of POU domain factors that are characterized by the presence of a highly conserved bipartite DNA binding domain, consisting of an amino-terminal specific subdomai...

  15. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  16. Allosteric Equilibria in the Binding of Fibrinogen to Platelets

    Science.gov (United States)

    de Cristofaro, Raimondo; Landolfi, Raffaele; de Candia, Erica; Castagnola, Massimo; di Cera, Enrico; Wyman, Jeffries

    1988-11-01

    The binding of fibrinogen to platelets occurs according to the law of mass action. The platelet receptor binds reversibly a single fibrinogen molecule and undergoes a conformational transition between two allosteric states, T and R, that differ in their affinity for fibrinogen. The equilibrium between the two forms is shifted by ADP toward the R (high-affinity) state, thus promoting the aggregation process. This model opens the way to consideration of allosteric modulation of the binding of fibrinogen to its platelet receptor.

  17. Agrobacterium rhizogenes mutants that fail to bind to plant cells.

    OpenAIRE

    Crews, J L; Colby, S; Matthysse, A G

    1990-01-01

    Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria ...

  18. Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

    Science.gov (United States)

    Lapelosa, Mauro; Gallicchio, Emilio; Levy, Ronald M.

    2011-01-01

    The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations. PMID:22368530

  19. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    Science.gov (United States)

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  20. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.

    2005-01-01

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site...

  1. A look at ligand binding thermodynamics in drug discovery.

    Science.gov (United States)

    Claveria-Gimeno, Rafael; Vega, Sonia; Abian, Olga; Velazquez-Campoy, Adrian

    2017-04-01

    Drug discovery is a challenging endeavor requiring the interplay of many different research areas. Gathering information on ligand binding thermodynamics may help considerably in reducing the risk within a high uncertainty scenario, allowing early rejection of flawed compounds and pushing forward optimal candidates. In particular, the free energy, the enthalpy, and the entropy of binding provide fundamental information on the intermolecular forces driving such interaction. Areas covered: The authors review the current status and recent developments in the application of ligand binding thermodynamics in drug discovery. The thermodynamic binding profile (Gibbs energy, enthalpy, and entropy of binding) can be used for lead selection and optimization (binding enthalpy, selectivity, and adaptability). Expert opinion: Binding thermodynamics provides fundamental information on the forces driving the formation of the drug-target complex. It has been widely accepted that binding thermodynamics may be used as a decision criterion along the ligand optimization process in drug discovery and development. In particular, the binding enthalpy may be used as a guide when selecting and optimizing compounds over a set of potential candidates. However, this has been recently called into question by arguing certain difficulties and in the light of certain experimental examples.

  2. Binding of ATP by pertussis toxin and isolated toxin subunits

    International Nuclear Information System (INIS)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

    1990-01-01

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

  3. Binding of ATP by pertussis toxin and isolated toxin subunits

    Energy Technology Data Exchange (ETDEWEB)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  4. Probing the binding of coumarins and cyclothialidines to DNA gyrase

    DEFF Research Database (Denmark)

    Kampranis, S C; Gormley, N A; Tranter, R

    1999-01-01

    B and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint......, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied...

  5. Regulation of inositol phospholipid binding and signaling through syndecan-4

    DEFF Research Database (Denmark)

    Couchman, John R; Vogt, Susan; Lim, Ssang-Taek

    2002-01-01

    inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen...... examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down...

  6. Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase.

    Science.gov (United States)

    Uni, Fumiya; Lee, Sunmi; Yatsunami, Rie; Fukui, Toshiaki; Nakamura, Satoshi

    2009-01-01

    Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

  7. Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 1: free energy of binding to a binding site.

    Science.gov (United States)

    Chunsrivirot, Surasak; Diao, Ying; Trout, Bernhardt L

    2011-10-18

    Crystallization is commonly used in a separation and purification process in the production of a wide range of materials in various industries. In industry, crystallization usually starts with heterogeneous nucleation on a foreign surface. The complicated mechanism of heterogeneous nucleation is not well understood; however, we hypothesize that there might be a possible correlation between binding affinity to a surface and enhancement of nucleation. Recent studies show that amorphous polymers can be used to control crystallization, selectively produce pharmaceutical polymorphs, and discover novel pharmaceutical polymorphs. To investigate the possible correlation between the binding affinity of one molecule to key binding sites (local binding) and heterogeneous nucleation activity as well as the possibility of using this binding affinity to help guide the selection of polymers that promote heterogeneous nucleation, we computed the free energy of binding of aspirin to four nonporous cross-linked polymers in an ethanol-water 38 v% mixture. These cross-linked polymers are poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS); all of them were cross-linked with divinylbenzene (DVB). These systems were used because their heterogeneous nucleation activities are available in literature, and the ranking is PAM > PCEA > PHBA ≈ PS. We generated three independent surfaces for each polymer and computed the free energy of binding of aspirin to the best binding site that we found on each surface. The average free energies of binding to the best sites of PAM, PCEA, PHBA, and PS are -20.4 ± 1.0, -16.7 ± 1.0, -14.4 ± 1.1, and -13.6 ± 1.1 kcal/mol, respectively. We found that the trend of the magnitudes of the average free energies of binding to the best sites is PAM > PCEA > PHBA ≈ PS. This trend is very similar to that of heterogeneous nucleation activity. Our results suggest the importance of the

  8. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A

    2015-01-01

    around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  9. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  10. Gentamicin binds to the megalin receptor as a competitive inhibitor using the common ligand binding motif of complement type repeats

    DEFF Research Database (Denmark)

    Dagil, Robert; O'Shea, Charlotte; Nykjær, Anders

    2013-01-01

    megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described...... to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists....

  11. RH421 binds into the ATP-binding site on the Na+/K+-ATPase.

    Science.gov (United States)

    Huličiak, Miroslav; Bazgier, Václav; Berka, Karel; Kubala, Martin

    2017-10-01

    The Na + /K + -ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na + /K + -ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na + /K + -ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Zinc induces structural reorganization of gelatin binding domain from human fibronectin and affects collagen binding.

    Science.gov (United States)

    Graille, Marc; Pagano, Maurice; Rose, Thierry; Ravaux, Michèle Reboud; van Tilbeurgh, Herman

    2010-06-09

    Fibronectin is a modular extracellular matrix protein involved in cell adhesion, cell motility, wound healing, and maintenance of cell morphology. It is composed of multiple repeats of three distinct modules: F(I), F(II), and F(III). Various combinations of these modules create fragments able to interact with different constituents of the extracellular matrix. Here, we present the 2.5-A resolution crystal structure of its 45-kDa gelatin-binding domain (GBD; 6F(I)-1F(II)-2F(II)-7F(I)-8F(I)-9F(I)), which also corresponds to the C-terminal half of the migration stimulating factor, a Fn splice variant expressed in human breast cancers. GBD forms a very compact zinc-mediated homodimer, in stark contrast with previous structures of fibronectin fragments. Most remarkably, 8F(I) no longer adopts the canonical F(I) fold but is composed of two long strands that associate with 7F(I) and 9F(I) into a large beta-sheet superdomain. Binding studies in solution confirmed that Zn induces conformational rearrangements and causes loss of binding of Fn-GBD to high-affinity collagen peptides. These data suggest the Zn may play a regulatory role for the cellular functions of fibronectin.

  13. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  14. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a cat......Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  15. Thermodynamic Characterization of New Positive Allosteric Modulators Binding to the Glutamate Receptor A2 Ligand-Binding Domain

    DEFF Research Database (Denmark)

    Nørholm, Ann-Beth; Francotte, Pierre; Goffin, Eric

    2014-01-01

    ,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven......, and combined with the shorter total simulation time, we found the OSP method to be more effective for this setup. Furthermore, from the molecular dynamics simulations, we extracted the enthalpies and entropies, and along with the ITC data, this suggested that the differences in binding free energies...

  16. Convulxin binds to native, human glycoprotein Ib alpha.

    Science.gov (United States)

    Kanaji, Sachiko; Kanaji, Taisuke; Furihata, Kenichi; Kato, Kazunobu; Ware, Jerry L; Kunicki, Thomas J

    2003-10-10

    Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

  17. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  18. Effects of heparin on insulin binding and biological activity

    International Nuclear Information System (INIS)

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-01-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125 I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells

  19. Principles for computational design of binding antibodies.

    Science.gov (United States)

    Baran, Dror; Pszolla, M Gabriele; Lapidoth, Gideon D; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D; Fleishman, Sarel J

    2017-10-10

    Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called "ideal" folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we ( i ) used sequence-design constraints derived from antibody multiple-sequence alignments, and ( ii ) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes.

  20. Development of radioimmunoassay for prolactin binding protein

    International Nuclear Information System (INIS)

    Raikar, R.S.; Sheth, A.R.

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10 10 M -1 . The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors. (author)

  1. Actin binding proteins, spermatid transport and spermiation*

    Science.gov (United States)

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  2. Interleukin-18 and interleukin-18 Binding Protein

    Directory of Open Access Journals (Sweden)

    Charles eDinarello

    2013-10-01

    Full Text Available Interleukin-18 (IL 18 is a member of the IL 1 family of cytokines. Increasing reports have expanded the role of IL 18 in mediating inflammation in animal models of disease using IL 18 deficient mice, neutralization of IL 18 or deficiency in the IL 18 receptor alpha chain. Similar to IL 1β, IL 18 is synthesized as an inactive precursor requiering processing by caspase 1 into an active cytokine but unlike IL 1β, the IL 18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL 18 is balanced by the presence of a high-affinity naturally occuring IL 18 binding protein (IL 18BP. In humans, disease increased disease severity can be associated with an imbalance of IL 18 to IL 18BP such that the levels of free IL 18 are elevated in the circulation. A role for IL 18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis and acute kidney injury, although in some diseases, IL 18 is protective. IL 18 plays a major role in the production of interferon-g from natural killer cells. The IL 18BP has been used safely in humans and clinical trials of IL 18BP as well as neutralizing anti-IL 18 antibodies are in clinical trials. This review updates the biology of IL 18 as well as its role in human disease

  3. Assessment Criteria of Bentonite Binding Properties

    Directory of Open Access Journals (Sweden)

    S. Żymankowska-Kumon

    2012-09-01

    Full Text Available The criteria, with which one should be guided at the assessment of the binding properties of bentonites used for moulding sands, areproposed in the paper. Apart from the standard parameter which is the active bentonite content, the unrestrained growth indicator should be taken into account since it seems to be more adequate in the estimation of the sand compression strength. The investigations performed for three kinds of bentonites, applied in the Polish foundry plants, subjected to a high temperature influences indicate, that the pathway of changes of the unrestrained growth indicator is very similar to the pathway of changes of the sand compression strength. Instead, the character of changes of the montmorillonite content in the sand in dependence of the temperature is quite different. The sand exhibits the significant active bentonite content, and the sand compression strength decreases rapidly. The montmorillonite content in bentonite samples was determined by the modern copper complex method of triethylenetetraamine (Cu(II-TET. Tests were performed for bentonites and for sands with those bentonites subjected to high temperatures influences in a range: 100-700ºC.

  4. Normas y valores: ¿Double bind?

    Directory of Open Access Journals (Sweden)

    Hjalmar Newmark

    2006-01-01

    Full Text Available La ponencia está dividida en dos panes. En la primera parte se propone, en primer lugar, describir qué significa para Luhmann el término globalización (1; en segundo lugar, se hace un bosquejo de la teoría luhmaniana del sistema político como sistema autorreferencial y autopoiético funcionalmente diferenciado de la sociedad moderna (2; y, por último, mostrar cómo el sistema de derecho cumple una función independiente del sistema político, pero, al mismo tiempo, se deja irritar por éste a través de su acoplamiento estructural (3. Y, en la segunda parte, se trata de mostrar (de manera suscinta cómo el sistema político, al cumplir con su función y, sobre todo, con la utilización del derecho, crea más conflictos de los que soluciona, en especial para la persona, al desdiferenciar los subsistemas funcionales y la sociedad misma (4 Palabras clave: Globalización, sistema politico, sistema jurídico, doub/e bind.

  5. Neptunium Binding Kinetics with Arsenazo(III)

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Leigh R. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Johnson, Aaron T. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Mezyk, Stephen P. [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2014-08-01

    This document has been prepared to meet FCR&D level 2 milestone M2FT-14IN0304021, “Report on the results of actinide binding kinetics with aqueous phase complexants” This work was carried out under the auspices of the Thermodynamics and Kinetics of Advanced Separations Systems FCR&D work package. The report details kinetics experiments that were performed to measure rates of aqueous phase complexation for pentavalent neptunium with the chromotropic dye Arsenazo III (AAIII). The studies performed were designed to determine how pH, ionic strength and AAIII concentration may affect the rate of the reaction. A brief comparison with hexavalent neptunium is also made. It was identified that as pH was increased the rate of reaction also increased, however increasing the ionic strength and concentration of AAIII had the opposite effect. Interestingly, the rate of reaction of Np(VI) with AAIII was found to be slower than that of the Np(V) reaction.

  6. Mouse models for core binding factor leukemia.

    Science.gov (United States)

    Chin, D W L; Watanabe-Okochi, N; Wang, C Q; Tergaonkar, V; Osato, M

    2015-10-01

    RUNX1 and CBFB are among the most frequently mutated genes in human leukemias. Genetic alterations such as chromosomal translocations, copy number variations and point mutations have been widely reported to result in the malfunction of RUNX transcription factors. Leukemias arising from such alterations in RUNX family genes are collectively termed core binding factor (CBF) leukemias. Although adult CBF leukemias generally are considered a favorable risk group as compared with other forms of acute myeloid leukemia, the 5-year survival rate remains low. An improved understanding of the molecular mechanism for CBF leukemia is imperative to uncover novel treatment options. Over the years, retroviral transduction-transplantation assays and transgenic, knockin and knockout mouse models alone or in combination with mutagenesis have been used to study the roles of RUNX alterations in leukemogenesis. Although successful in inducing leukemia, the existing assays and models possess many inherent limitations. A CBF leukemia model which induces leukemia with complete penetrance and short latency would be ideal as a platform for drug discovery. Here, we summarize the currently available mouse models which have been utilized to study CBF leukemias, discuss the advantages and limitations of individual experimental systems, and propose suggestions for improvements of mouse models.

  7. Temporal binding within and across events

    Science.gov (United States)

    DuBrow, Sarah; Davachi, Lila

    2016-01-01

    Remembering the order in which events occur is a fundamental component of episodic memory. However, the neural mechanisms supporting serial recall remain unclear. Behaviorally, serial recall is greater for information encountered within the same event compared to across event boundaries, raising the possibility that contextual stability may modulate the cognitive and neural processes supporting serial encoding. In the present study, we used fMRI during the encoding of consecutive face and object stimuli to elucidate the neural encoding signatures supporting subsequent serial recall behavior both within and across events. We found that univariate BOLD activation in both the middle hippocampus and left ventrolateral prefrontal cortex (PFC) was associated with subsequent serial recall of items that occur across event boundaries. By contrast, successful serial encoding within events was associated with increased functional connectivity between the hippocampus and ventromedial PFC, but not with univariate activation in these or other regions. These findings build on evidence implicating hippocampal and PFC processes in encoding temporal aspects of memory. They further suggest that these encoding processes are influenced by whether binding occurs within a stable context or bridges two adjacent but distinct events. PMID:27422018

  8. Where does mediator bind in vivo?

    Science.gov (United States)

    Fan, Xiaochun; Struhl, Kevin

    2009-01-01

    The Mediator complex associates with RNA polymerase (Pol) II, and it is recruited to enhancer regions by activator proteins under appropriate environmental conditions. However, the issue of Mediator association in yeast cells is controversial. Under optimal growth conditions (YPD medium), we were unable to detect Mediator at essentially any S. cerevisiae promoter region, including those supporting very high levels of transcription. In contrast, whole genome microarray experiments in synthetic complete (SC) medium reported that Mediator associates with many genes at both promoter and coding regions. As assayed by chromatin immunoprecipitation, we show that there are a small number of Mediator targets in SC medium that are not observed in YPD medium. However, most Mediator targets identified in the genome-wide analysis are false positives that arose for several interrelated reasons: the use of overly lenient cut-offs; artifactual differences in apparent IP efficiencies among different genomic regions in the untagged strain; low fold-enrichments making it difficult to distinguish true Mediator targets from false positives that occur in the absence of the tagged Mediator protein. Lastly, apparent Mediator association in highly active coding regions is due to a non-specific effect on accessibility due to the lack of nucleosomes, not to a specific association of Mediator. These results indicate that Mediator does not bind to numerous sites in the yeast genome, but rather selectively associates with a limited number of upstream promoter regions in an activator- and stress-specific manner.

  9. Where does mediator bind in vivo?

    Directory of Open Access Journals (Sweden)

    Xiaochun Fan

    Full Text Available The Mediator complex associates with RNA polymerase (Pol II, and it is recruited to enhancer regions by activator proteins under appropriate environmental conditions. However, the issue of Mediator association in yeast cells is controversial. Under optimal growth conditions (YPD medium, we were unable to detect Mediator at essentially any S. cerevisiae promoter region, including those supporting very high levels of transcription. In contrast, whole genome microarray experiments in synthetic complete (SC medium reported that Mediator associates with many genes at both promoter and coding regions.As assayed by chromatin immunoprecipitation, we show that there are a small number of Mediator targets in SC medium that are not observed in YPD medium. However, most Mediator targets identified in the genome-wide analysis are false positives that arose for several interrelated reasons: the use of overly lenient cut-offs; artifactual differences in apparent IP efficiencies among different genomic regions in the untagged strain; low fold-enrichments making it difficult to distinguish true Mediator targets from false positives that occur in the absence of the tagged Mediator protein. Lastly, apparent Mediator association in highly active coding regions is due to a non-specific effect on accessibility due to the lack of nucleosomes, not to a specific association of Mediator.These results indicate that Mediator does not bind to numerous sites in the yeast genome, but rather selectively associates with a limited number of upstream promoter regions in an activator- and stress-specific manner.

  10. IGF Binding Protein-5 Induces Cell Senescence

    Directory of Open Access Journals (Sweden)

    Fumihiro Sanada

    2018-02-01

    Full Text Available Cellular senescence is the complex process of deterioration that drives the aging of an organism, resulting in the progressive loss of organ function and eventually phenotypic aging. Senescent cells undergo irreversible growth arrest, usually by inducing telomere shortening. Alternatively, senescence may also occur prematurely in response to various stress stimuli, such as oxidative stress, DNA damage, or activated oncogenes. Recently, it has been shown that IGF binding protein-5 (IGFBP-5 with the induction of the tumor suppressor p53 is upregulated during cellular senescence. This mechanism mediates interleukin-6/gp130-induced premature senescence in human fibroblasts, irradiation-induced premature senescence in human endothelial cells (ECs, and replicative senescence in human ECs independent of insulin-like growth factor I (IGF-I and IGF-II. Additionally, a link between IGFBP-5, hyper-coagulation, and inflammation, which occur with age, has been implicated. Thus, IGFBP-5 seems to play decisive roles in controlling cell senescence and cell inflammation. In this review, we describe the accumulating evidence for this role of IGFBP-5 including our new finding.

  11. FTIR spectroscopy of flavin-binding photoreceptors.

    Science.gov (United States)

    Yamada, Daichi; Kandori, Hideki

    2014-01-01

    Light-induced difference Fourier transform infrared (FTIR) spectroscopy is a powerful, sensitive, and informative method to study structure-function relationships in photoreceptive proteins. Strong absorption of water in the IR region is always problematic in this method, but if water content in the sample is controlled during measurements, this method can provide useful information on a single protein-bound water molecule. We established three kinds of sample preparations: hydrated film, redissolved sample, and concentrated solution. Hydrated films were used for the measurements of LOV and BLUF domains, where accurate difference FTIR spectra were obtained in the whole mid-IR region (4,000-800 cm(-1)). Vibrations of S-H stretch of cysteine, O-H stretch of water, and O-H stretch of tyrosine provide important information on hydrogen bonds in these proteins. Redissolved samples were used for the measurements of (6-4) photolyase, in which enzymatic turnover takes place. From the illumination time-dependence of excess amount of substrate, it is possible to isolate the signal originating from the binding of enzyme to substrate. If proteins are less tolerant to drying, as for example cryptochromes of the DASH type, concentrated solution is used. Detailed methodological aspects in light-induced difference FTIR spectroscopy are reviewed by mainly focusing on our results.

  12. Integrating structural and mutagenesis data to elucidate GPCR ligand binding

    DEFF Research Database (Denmark)

    Munk, Christian; Harpsøe, Kasper; Hauser, Alexander S

    2016-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of human membrane proteins, as well as drug targets. A recent boom in GPCR structural biology has provided detailed images of receptor ligand binding sites and interactions on the molecular level. An ever-increasing number of ligands...... elucidate new GPCR ligand binding sites, and ultimately design drugs with tailored pharmacological activity....

  13. Perturbation method for calculating impurity binding energy in an ...

    Indian Academy of Sciences (India)

    Perturbation method is used to calculate the binding energy within the framework of effective mass approximation and taking into account the effect of dielectric mismatch between the dot and the barrier material. The ground-state binding energy of the donor is computed as a function of dot size for finite confinement.

  14. Spectral characterization and DNA binding properties of lanthanide(III)

    African Journals Online (AJOL)

    Spectral data of complexes suggest that the ligand binds metal ion through pyridine- nitrogen, azomethine-nitrogen and amido-oxygen donor atoms. Electrochemical behaviour of metal complexes was investigated by using cyclic voltammetry. The complexes undergo quasi-reversible one electron reduction. The binding ...

  15. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  16. Synthesis, characterization, crystal structure and DNA-binding study ...

    Indian Academy of Sciences (India)

    BOLIN

    The results suggest that neutral complexes 2a and 2b bind to DNA in an intercalative mode. On the other hand, cationic complexes 1a and 1b interact with DNA via weak intercalative or groove binding mode. (NOTE: See more examples of Graphical Abstracts in Journal website, http://www.ias.ac.in/chemsci/index.html under ...

  17. Modeling Shear Induced Von Willebrand Factor Binding to Collagen

    Science.gov (United States)

    Dong, Chuqiao; Wei, Wei; Morabito, Michael; Webb, Edmund; Oztekin, Alparslan; Zhang, Xiaohui; Cheng, Xuanhong

    2017-11-01

    Von Willebrand factor (vWF) is a blood glycoprotein that binds with platelets and collagen on injured vessel surfaces to form clots. VWF bioactivity is shear flow induced: at low shear, binding between VWF and other biological entities is suppressed; for high shear rate conditions - as are found near arterial injury sites - VWF elongates, activating its binding with platelets and collagen. Based on parameters derived from single molecule force spectroscopy experiments, we developed a coarse-grain molecular model to simulate bond formation probability as a function of shear rate. By introducing a binding criterion that depends on the conformation of a sub-monomer molecular feature of our model, the model predicts shear-induced binding, even for conditions where binding is highly energetically favorable. We further investigate the influence of various model parameters on the ability to predict shear-induced binding (vWF length, collagen site density and distribution, binding energy landscape, and slip/catch bond length) and demonstrate parameter ranges where the model provides good agreement with existing experimental data. Our results may be important for understanding vWF activity and also for achieving targeted drug therapy via biomimetic synthetic molecules. National Science Foundation (NSF),Division of Mathematical Sciences (DMS).

  18. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

    2017-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherentl...

  19. Mannan-binding lectin in astma and allergy

    DEFF Research Database (Denmark)

    Kaur, S.; Thiel, Steffen; Sarma, P.U.

    2006-01-01

    Mannan-binding lectin (MBL) is a vital and versatile component of innate immunity. It is present in serum and may bind to a plethora of microbial pathogens and mediate opsonization of these by complement-dependent and/or independent mechanisms. Low-MBL levels in serum, attributed to certain genet...... of MBL in asthma and allergy...

  20. Metal ion binding with dehydroannulenes – Plausible two ...

    Indian Academy of Sciences (India)

    WINTEC

    Abstract. Theoretical investigations have been carried out at B3LYP/6-311++G** level of theory to study the binding interaction of various metal ions, Li+, Na+ and K+ with dehydroannulene systems. The present study reveals that alkali metal ions bind strongly to dehydroannulenes and the passage through the central.

  1. Heat Shock Protein 27 Inhibits Apoptosis by Binding Cytochrome C

    National Research Council Canada - National Science Library

    Carper, Stephen

    2002-01-01

    ...) and cytochrome c in the inhibition of apoptosis. The scope of the study will include: measuring the binding of hsp27to cytochrome c in vivo, determining why hsp27 binds to cytochrome c and determining the fate of the hsp27...

  2. Heat Shock Protein 27 Inhibits Apoptosis by Binding Cytochrome c

    National Research Council Canada - National Science Library

    Carper, Stephen

    2003-01-01

    ...) and cytochrome c in the inhibition of apoptosis. The scope of the study was to include: measuring the binding of hsp27 to cytochrome c in vivo, determining why hsp27 binds to cytochrome c and determining the fate of the hsp27...

  3. Comparing mannose binding lectin genetic diversity in intracellular ...

    African Journals Online (AJOL)

    SERVER

    2007-09-05

    Sep 5, 2007 ... binding lectin of Escherichia coli (Kawasaki et al., 1989) and Salmonella (Ihara et al., 1991). However some reports could not find any effect of mannose binding lectin on complement activation upon extracellular infec- tion of Staphylococcus aureus (Cunion et al., 2001). In intracellular infections, there is ...

  4. Extrapolations of nuclear binding energies from new linear mass relations

    DEFF Research Database (Denmark)

    Hove, D.; Jensen, A. S.; Riisager, K.

    2013-01-01

    We present a method to extrapolate nuclear binding energies from known values for neighboring nuclei. We select four specific mass relations constructed to eliminate smooth variation of the binding energy as function nucleon numbers. The fast odd-even variations are avoided by comparing nuclei...

  5. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  6. Binding of reactive organophosphate by oximes via hydrogen bond

    Indian Academy of Sciences (India)

    In this contribution, the ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments confirm that oximes bind the organophosphate ...

  7. Binding-Induced Fluorescence of Serotonin Transporter Ligands

    DEFF Research Database (Denmark)

    Wilson, James; Ladefoged, Lucy Kate; Babinchak, Michael

    2014-01-01

    The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP(+)) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP(+)) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP(+)), has...

  8. Modification of opiate agonist binding by pertussis toxin

    Energy Technology Data Exchange (ETDEWEB)

    Abood, M.E.; Lee, N.M.; Loh, H.H.

    1986-03-05

    Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin. This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.

  9. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  10. Biosensors engineered from conditionally stable ligand-binding domains

    Energy Technology Data Exchange (ETDEWEB)

    Church, George M.; Feng, Justin; Mandell, Daniel J.; Baker, David; Fields, Stanley; Jester, Benjamin Ward; Tinberg, Christine Elaine

    2017-09-19

    Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

  11. Identifying the binding mode of a molecular scaffold

    Science.gov (United States)

    Chema, Doron; Eren, Doron; Yayon, Avner; Goldblum, Amiram; Zaliani, Andrea

    2004-01-01

    We describe a method for docking of a scaffold-based series and present its advantages over docking of individual ligands, for determining the binding mode of a molecular scaffold in a binding site. The method has been applied to eight different scaffolds of protein kinase inhibitors (PKI). A single analog of each of these eight scaffolds was previously crystallized with different protein kinases. We have used FlexX to dock a set of molecules that share the same scaffold, rather than docking a single molecule. The main mode of binding is determined by the mode of binding of the largest cluster among the docked molecules that share a scaffold. Clustering is based on our `nearest single neighbor' method [J. Chem. Inf. Comput. Sci., 43 (2003) 208-217]. Additional criteria are applied in those cases in which more than one significant binding mode is found. Using the proposed method, most of the crystallographic binding modes of these scaffolds were reconstructed. Alternative modes, that have not been detected yet by experiments, could also be identified. The method was applied to predict the binding mode of an additional molecular scaffold that was not yet reported and the predicted binding mode has been found to be very similar to experimental results for a closely related scaffold. We suggest that this approach be used as a virtual screening tool for scaffold-based design processes.

  12. Partial association of restriction polymorphism of the ligand binding ...

    African Journals Online (AJOL)

    Partial association of restriction polymorphism of the ligand binding domain of human androgen receptor in prostate cancer. ... Background: Human androgen receptor (AR) functions as a steroid-hormone activated transcription factor. The receptor binds to its ligand (testosterone or dihydrotestosterone) and is translocated to ...

  13. Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Duijn, van E.; Barendregt, A.; Dyer, K.; Tainer, J.A.; Stoltenburg, R.; Strehlitz, B.; Levisson, M.; Smidt, H.; Oost, van der J.

    2012-01-01

    Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated

  14. Inhibition of nuclear T3 binding by fatty acids

    NARCIS (Netherlands)

    Wiersinga, W. M.; Chopra, I. J.; Teco, G. N.

    1988-01-01

    Studies were performed to evaluate a possible modulatory role of lipids on the binding of T3 to rat liver nuclear receptors in vitro. Unsaturated fatty acids were potent inhibitors of the binding of [125I] T3 to isolated rat liver nuclei. Doses (in mumol/L) causing a 50% inhibition of nuclear T3

  15. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Support Vector Machine (SVM) is a state-of-the-art classifica- tion technique. Using canonical binding model, the C2H2 zinc finger protein–DNA interaction interface is modelled by the pairwise amino acid–base interactions. Using a classification framework, known examples of non-binding ZF–DNA pairs.

  16. Modification of opiate agonist binding by pertussis toxin

    International Nuclear Information System (INIS)

    Abood, M.E.; Lee, N.M.; Loh, H.H.

    1986-01-01

    Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in 3 (H)-DADLE binding as compared with membranes treated identically without toxin. This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding

  17. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Incorporating evolution of transcription factor binding sites into annotated alignments. 841. J. Biosci. 32(5), August 2007. 1. Introduction. A majority of computational approaches that aim to predict transcription factor binding sites employ cross- species comparison to focus on conserved locations. Such a comparison helps in ...

  18. Fluorescence spectroscopic studies on binding of a flavonoid ...

    Indian Academy of Sciences (India)

    Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime ...

  19. Perturbation method for calculating impurity binding energy in an ...

    Indian Academy of Sciences (India)

    Nilanjan Sil

    2017-12-18

    Dec 18, 2017 ... Abstract. In the present paper, we have studied the binding energy of the shallow donor hydrogenic impurity, which is confined in an inhomogeneous cylindrical quantum dot (CQD) of GaAs-AlxGa1−xAs. Perturbation method is used to calculate the binding energy within the framework of effective mass ...

  20. Synthesis, DNA binding and cytotoxic evaluation of aminoquinoline ...

    Indian Academy of Sciences (India)

    DNA binding studies of selected isomeric compounds showed interaction withDNA via intercalation mode with higher binding affinity of 4-substituted quinolines rather than 2-substituted counterparts. Further, all compounds were screened for cytotoxic activity against three human cancer cell lines,among them compound 2c ...