WorldWideScience

Sample records for bacterial enhancer binding

  1. A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

    Science.gov (United States)

    Bowman, William C.; Kranz, Robert G.

    1998-01-01

    A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called ς54 or ςN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/ς54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/ς70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed. PMID:9637689

  2. A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

    OpenAIRE

    Bowman, William C.; Kranz, Robert G.

    1998-01-01

    A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the D...

  3. CCAAT/enhancer-binding protein δ facilitates bacterial dissemination during pneumococcal pneumonia in a platelet-activating factor receptor-dependent manner

    OpenAIRE

    Duitman, JanWillem; Schouten, Marcel; Groot, Angelique P.; Borensztajn, Keren S.; Daalhuisen, Joost B.; Florquin, Sandrine; van der Poll, Tom; Spek, C Arnold

    2012-01-01

    CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ−/− mice are relatively resistant to pneumococcal pneumonia, as ind...

  4. Capsular Sialic Acid of Streptococcus suis Serotype 2 Binds to Swine Influenza Virus and Enhances Bacterial Interactions with Virus-Infected Tracheal Epithelial Cells

    Science.gov (United States)

    Wang, Yingchao; Gagnon, Carl A.; Savard, Christian; Music, Nedzad; Srednik, Mariela; Segura, Mariela; Lachance, Claude; Bellehumeur, Christian

    2013-01-01

    Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model. PMID:24082069

  5. Structural basis for entropy-driven cellulose binding by a type-A cellulose-binding module (CBM) and bacterial expansin

    OpenAIRE

    Georgelis, Nikolaos; Yennawar, Neela H.; Cosgrove, Daniel J.

    2012-01-01

    Components of modular cellulases, type-A cellulose-binding modules (CBMs) bind to crystalline cellulose and enhance enzyme effectiveness, but structural details of the interaction are uncertain. We analyzed cellulose binding by EXLX1, a bacterial expansin with ability to loosen plant cell walls and whose domain D2 has type-A CBM characteristics. EXLX1 strongly binds to crystalline cellulose via D2, whereas its affinity for soluble cellooligosaccharides is weak. Calorimetry indicated cellulose...

  6. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    OpenAIRE

    Frick, I M; Wikström, M.; Forsén, S.; Drakenberg, T; Gomi, H.; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide,...

  7. Bacterial Enhancement of Vinyl Fouling by Algae

    OpenAIRE

    Holmes, Paul E.

    1986-01-01

    The role of bacteria in the development of algae on low-density vinyl was investigated. Unidentified bacterial contaminants in unialgal stock cultures of Phormidium faveolarum and Pleurochloris pyrenoidosa enhanced, by 1 to 2 orders of magnitude, colonization of vinyl by these algae, as determined by epifluorescence microscopy counts and chlorophyll a in extracts of colonized vinyl. Colonization by bacteria always preceded that by algae. Scanning electron microscopy of the colonized Phormidiu...

  8. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  9. Bacterial binding to extracellular proteins - in vitro adhesion

    DEFF Research Database (Denmark)

    Schou, C.; Fiehn, N.-E.

    1999-01-01

    Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

  10. Dispersal networks for enhancing bacterial degradation in heterogeneous environments

    Energy Technology Data Exchange (ETDEWEB)

    Banitz, Thomas, E-mail: thomas.banitz@ufz.de [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Wick, Lukas Y.; Fetzer, Ingo [Department of Environmental Microbiology, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Frank, Karin [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Harms, Hauke [Department of Environmental Microbiology, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Johst, Karin [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany)

    2011-10-15

    Successful biodegradation of organic soil pollutants depends on their bioavailability to catabolically active microorganisms. In particular, environmental heterogeneities often limit bacterial access to pollutants. Experimental and modelling studies revealed that fungal networks can facilitate bacterial dispersal and may thereby improve pollutant bioavailability. Here, we investigate the influence of such bacterial dispersal networks on biodegradation performance under spatially heterogeneous abiotic conditions using a process-based simulation model. To match typical situations in polluted soils, two types of abiotic conditions are studied: heterogeneous bacterial dispersal conditions and heterogeneous initial resource distributions. The model predicts that networks facilitating bacterial dispersal can enhance biodegradation performance for a wide range of these conditions. Additionally, the time horizon over which this performance is assessed and the network's spatial configuration are key factors determining the degree of biodegradation improvement. Our results support the idea of stimulating the establishment of fungal mycelia for enhanced bioremediation of polluted soils. - Highlights: > Bacterial dispersal networks can considerably improve biodegradation performance. > They facilitate bacterial access to dispersal-limited areas and remote resources. > Abiotic conditions, time horizon and network structure govern the improvements. > Stimulating the establishment of fungal mycelia promises enhanced soil remediation. - Simulation modelling demonstrates that fungus-mediated bacterial dispersal can considerably improve the bioavailability of organic pollutants under spatially heterogeneous abiotic conditions typical for water-unsaturated soils.

  11. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    Science.gov (United States)

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  12. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  13. STD NMR spectroscopy: a case study of fosfomycin binding interactions in living bacterial cells

    Energy Technology Data Exchange (ETDEWEB)

    Milagre, Cintia D.F.; Cabeca, Luis Fernando; Martins, Lucas G.; Marsaioli, Anita J., E-mail: anita@iq [Universidade Estadual de Campinas (IQ/UNICAMP), SP (Brazil). Inst. de Quimica

    2011-07-01

    A saturation transfer difference (STD) NMR experiment was successfully employed to observe the binding interactions of fosfomycin resistant and non-resistant bacterial strains using living cell suspensions, without the need for isotopic labelling of the ligand or receptor. (author)

  14. Binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1976-01-01

    Bacterial transformation in relation to DNA transport and competence in Streptococcus pneumoniae (also called Diplococcus pneumoniae) is discussed. This species will serve as a model with which to compare transformation in other bacterial species, particularly Bacillus subtilis and Haemophilus influenzae, with emphasis on the many similarities as well as differences.

  15. Using synthetic bacterial enhancers to reveal a looping-based mechanism for quenching-like repression

    Science.gov (United States)

    Brunwasser-Meirom, Michal; Pollak, Yaroslav; Goldberg, Sarah; Levy, Lior; Atar, Orna; Amit, Roee

    2016-02-01

    We explore a model for `quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10-11 bp insertions or deletions (INDELs) and sensitive to 5-6 bp INDELs. We test this prediction on 61 σ54-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat.

  16. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  17. Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD

    OpenAIRE

    Andrade, Fábia K; Moreira, Susana Margarida Gomes; Domingues, Lucília; Gama, F. M.

    2010-01-01

    The attachment of cells to biomedical materials can be improved by using adhesion sequences, such as Arg-Gly-Asp (RGD), found in several extracellular matrix proteins. In this work, bifunctional recombinant proteins, with a Cellulose-Binding Module (CBM), from the cellulosome of Clostridium thermocellum and cell binding sequences - RGD, GRGDY - were cloned and expressed in E.coli. These RGD-containing cellulose binding proteins were purified and used to coat bacterial cellulose fibres. Its ef...

  18. 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...

  19. 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.

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

    International Nuclear Information System (INIS)

    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

  1. The Bacterial Translocon SecYEG Opens upon Ribosome Binding*

    OpenAIRE

    Knyazev, Denis G.; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-01-01

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which ha...

  2. The bacterial translocon SecYEG opens upon ribosome binding.

    Science.gov (United States)

    Knyazev, Denis G; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-06-21

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  3. The Bacterial Translocon SecYEG Opens upon Ribosome Binding*

    Science.gov (United States)

    Knyazev, Denis G.; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-01-01

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  4. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  5. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    International Nuclear Information System (INIS)

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 (angstrom) above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational

  6. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

    Science.gov (United States)

    Singh, Satinder K; Yamashita, Atsuko; Gouaux, Eric

    2007-08-23

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of

  7. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

    Directory of Open Access Journals (Sweden)

    Xiaofei Gao

    2009-12-01

    Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

  8. A method for in vivo identification of bacterial small RNA-binding proteins.

    Science.gov (United States)

    Osborne, Jonathan; Djapgne, Louise; Tran, Bao Quoc; Goo, Young Ah; Oglesby-Sherrouse, Amanda G

    2014-12-01

    Small bacterial regulatory RNAs (sRNAs) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA-binding protein. However, not all sRNAs rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNAs. Here, we describe a methodology for identifying in vivo-binding proteins of sRNAs, developed using the iron-responsive PrrF and PrrH sRNAs of Pseudomonas aeruginosa. RNA was isolated from iron-depleted cultures, which were irradiated to cross-link nucleoprotein complexes. Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF- and PrrH-binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo. Hfq binding to the PrrF and PrrH sRNAs was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa. As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

  9. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  10. Proteolytic activation of human pancreatitis associated protein is required for peptidoglycan binding and bacterial aggregation

    OpenAIRE

    Medveczky, Péter; Szmola, Richárd; Sahin-Tóth, Miklós

    2009-01-01

    Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antib...

  11. Vitamin C enhances bacterial cellulose production in Gluconacetobacter xylinus.

    Science.gov (United States)

    Keshk, Sherif M A S

    2014-01-01

    Influence of vitamin C (ascorbic acid) on bacterial cellulose (BC) production and crystal structure was studied using four strains of Gluconacetobacter xylinus (ATCC 10245, IFO 13693, 13772 and 13773). BC productivity of all strains was increased in presence of vitamin C (0.5% w/w), the average BC production reached 0.47 g/30 ml compared with 0.25 g/30 ml without vitamin C. Enhanced productivity is associated with a decrease in gluconic acid concentration that is produced from Gluconacetobacter xylinus during BC production. X-ray results showed that the crystallinity index of BC produced in presence of ascorbic acid was the lowest with remarkable change in d-spacing. These results were confirmed by using solid state (13)CNMR. The increase in BC yield in presence of vitamin C is due to its antioxidant behavior and confirms our past work on lignosulfonate influence on BC.

  12. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  13. Impaired neutrophil function in 24p3 null mice contributes to enhanced susceptibility to bacterial infections

    OpenAIRE

    Liu, Zhuoming; Petersen, Robert; Devireddy, L.

    2013-01-01

    Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (...

  14. Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

    Science.gov (United States)

    Rumyantsev, Konstantin A.; Shcherbakova, Daria M.; Zakharova, Natalia I.; Emelyanov, Alexander V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2015-12-01

    Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

  15. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Annamari Paino

    Full Text Available Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI, was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control

  16. Human tandem-repeat-type galectins bind bacterial non-βGal polysaccharides

    DEFF Research Database (Denmark)

    Knirel, Yu A.; Gabius, H.-J.; Blixt, Klas Ola;

    2014-01-01

    ), prompted us to establish an array with bacterial polysaccharides. We addressed the question whether sugar determinants other than β-galactosides may be docking sites, using human galectins-4, -8, and -9. Positive controls with histo-blood group ABH-epitopes and the E. coli 086 polysaccharide ascertained...... the suitability of the set-up. Significant signal generation, depending on type of galectin and polysacchride, was obtained. Presence of cognate β-galactoside-related epitopes within a polysaccharide chain or its branch will not automatically establish binding properties, and structural constellations lacking...

  17. Pillar[5]arene-Based Glycoclusters: Synthesis and Multivalent Binding to Pathogenic Bacterial Lectins.

    Science.gov (United States)

    Buffet, Kevin; Nierengarten, Iwona; Galanos, Nicolas; Gillon, Emilie; Holler, Michel; Imberty, Anne; Matthews, Susan E; Vidal, Sébastien; Vincent, Stéphane P; Nierengarten, Jean-François

    2016-02-24

    The synthesis of pillar[5]arene-based glycoclusters has been readily achieved by CuAAC conjugations of azido- and alkyne-functionalized precursors. The lectin binding properties of the resulting glycosylated multivalent ligands have been studied by at least two complementary techniques to provide a good understanding. Three lectins were selected from bacterial pathogens based on their potential therapeutic applications as anti-adhesives, namely LecA and LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria. As a general trend, multivalency improved the binding to lectins and a higher affinity can be obtained by increasing to a certain limit the length of the spacer arm between the carbohydrate subunits and the central macrocyclic core.

  18. Engineered Bacterial Metal-binding Proteins for Nanoscale Self-assembly and heavy Metal Tolerance

    Science.gov (United States)

    Hall Sedlak, Ruth Amanda

    Implementing biological principles in material synthesis and assembly is one way to expand our abilities to efficiently assemble nanoscale materials and devices. Specifically, recent advances in identifying peptides that bind inorganic materials with high affinity and specificity has spurred investigation of protein models for nanoscale inorganic assembly. This dissertation presents the results of my studies of several E. coli proteins engineered to bind inorganic materials through simple peptide motifs. I demonstrate that these proteins modulate the self-assembly of DNA-based nanostructures and can introduce heavy metal tolerance into metal-sensitive bacteria. Chapter 2 explores use of the engineered F plasmid DNA relaxase/helicase TraI for the self-assembly of complex DNA-protein-gold nanostructures. The full-length protein is engineered with a gold binding motif at an internal permissive site (TraI369GBP1-7x), while a truncated version of TraI is engineered with the same gold binding motif at the C-terminus (TraI361GBP1-7x). Both constructs bind gold nanoparticles while maintaining their DNA binding activity, and transmission electron microscopy reveals TraI369GBP1-7x utilizes its non-specific DNA binding activity to decorate single-stranded and double-stranded DNA with gold nanoparticles. The self assembly principles demonstrated in this work will be fundamental to constructing higher ordered hybrid nanostructures through DNA-protein-nanoparticle interactions. Chapter 3 studies the effects of expressing inorganic binding peptides within cells. I identified a silver binding peptide that, when fused to the periplasmic maltose binding protein, protects E. coli from silver toxicity in batch culture and reduces silver ions to silver nanoparticles within the bacterial periplasm. Engineered metal-ion tolerant microorganisms such as this E. coli could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo

  19. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    LENUS (Irish Health Repository)

    Jones, Robert T

    2010-05-12

    Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

  20. Photorhabdus adhesion modification protein (Pam binds extracellular polysaccharide and alters bacterial attachment

    Directory of Open Access Journals (Sweden)

    Joyce Susan A

    2010-05-01

    Full Text Available Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C and human (37°C temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect

  1. Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS*

    Science.gov (United States)

    Yan, Robert; Konarev, Petr V.; Iannuzzi, Clara; Adinolfi, Salvatore; Roche, Béatrice; Kelly, Geoff; Simon, Léa; Martin, Stephen R.; Py, Béatrice; Barras, Frédéric; Svergun, Dmitri I.; Pastore, Annalisa

    2013-01-01

    The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly. PMID:23839945

  2. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins.

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

    Directory of Open Access Journals (Sweden)

    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. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

  5. Microbial interactions chapter: binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Genetic transformation of bacteria by DNA released from cells of a related strain is discussed. The mechanism by which the giant information-bearing molecules of DNA are transported into the bacterial cell was investigated. It was concluded that the overall process of DNA uptake consists of two main steps, binding of donor DNA to the outside of the cell and entry of the bound DNA into the cell. Each step is discussed in detail. Inasmuch as these phenomena occur at the cell surface, they are related to structures and functions of the cell wall and membrane. In addition, the development of competence, that is the formation of cell surface structures allowing DNA uptake, is examined from both a physiological and evolutionary point of view. Genetic transfer mediated by free DNA is an obvious and important form of cellular interaction. The development of competence involves another, quite distinct system of interaction between bacterial cells. Streptococcus pneumoniae, Bacillus subtilis, and Hemophilus influenzae were used as the test organisms. 259 references.

  6. Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

    Science.gov (United States)

    Boiteux, Céline; Vorobyov, Igor; French, Robert J; French, Christopher; Yarov-Yarovoy, Vladimir; Allen, Toby W

    2014-09-01

    Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors. PMID:25136136

  7. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  8. Deleted in Malignant Brain Tumors 1 is up-regulated in bacterial endocarditis and binds to components of vegetations

    DEFF Research Database (Denmark)

    Müller, Hanna; Renner, Marcus; Helmke, Burkhard M;

    2009-01-01

    OBJECTIVE: Bacterial endocarditis is a frequent infectious cardiac disease, especially in patients with congenital or acquired heart defects. It is characterized by bacterial colonization of the heart valves and the appearance of vegetations consisting of fibrin, blood cells, and bacteria...... be linked to the development of vegetations. METHODS: Heart tissue of 19 patients with bacterial endocarditis and 10 controls without bacterial endocarditis was analyzed by immunohistochemistry. The effect of human recombinant Deleted in Malignant Brain Tumors 1 on erythrocyte aggregation was measured using...... an automated red blood cell aggregometer MA1. Binding of human recombinant Deleted in Malignant Brain Tumors 1 to erythrocyte membranes, platelets, fibrin, and fibrinogen was analyzed by Western blotting and enzyme-linked immunosorbent assay. RESULTS: Deleted in Malignant Brain Tumors 1 expression was up...

  9. Enhancement of mouse sperm motility by trophinin-binding peptide

    Directory of Open Access Journals (Sweden)

    Park Seong

    2012-11-01

    Full Text Available Abstract Background Trophinin is an intrinsic membrane protein that forms a complex in the cytoplasm with bystin and tastin, linking it microtubule-associated motor dynein (ATPase in some cell types. Previously, we found that human sperm tails contain trophinin, bystin and tastin proteins, and that trophinin-binding GWRQ (glycine, tryptophan, arginine, glutamine peptide enhanced motility of human sperm. Methods Immunohistochemistry was employed to determine trophinin protein in mouse spermatozoa from wild type mouse, by using spermatozoa from trophinin null mutant mice as a negative control. Multivalent 8-branched GWRQ (glycine, tryptophan, arginine, glutamine peptide or GWRQ-MAPS, was chemically synthesized, purified by HPLC and its structure was confirmed by MALDI-TOF mass spectrometry. Effect of GWRQ-MAPS on mouse spermatozoa from wild type and trophinin null mutant was assessed by a computer-assisted semen analyzer (CASA. Results Anti-trophinin antibody stained the principal (central piece of the tail of wild type mouse sperm, whereas the antibody showed no staining on trophinin null sperm. Phage particles displaying GWRQ bound to the principal piece of sperm tail from wild type but not trophinin null mice. GWRQ-MAPS enhanced motility of spermatozoa from wild type but not trophinin null mice. CASA showed that GWRQ-MAPS enhanced both progressive motility and rapid motility in wild type mouse sperm. Conclusions Present study established the expression of trophinin in the mouse sperm tail and trophinin-dependent effect of GWRQ-MAPS on sperm motility. GWRQ causes a significant increase in sperm motility.

  10. Biointerface: protein enhanced stem cells binding to implant surface.

    Science.gov (United States)

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

  11. Enhanced Activity of Topical Hydrocortisone by Competitive Binding of Corticosteroid-Binding Globulin.

    Science.gov (United States)

    Bodor, Erik T; Wu, Whei-Mei; Chandran, V Ravi; Bodor, Nicholas

    2016-09-01

    Atopic dermatitis of sensitive areas such as the face, particularly in children, is a difficult disease to treat as the standard therapeutic, topical steroids, is contraindicated for this application in children. Hydrocortisone (HC) can be used in these instances because it has been shown to be safe, but is often ineffective as it is a relatively weak steroid, especially at over-the-counter concentrations. To enhance the local topical activity of HC, the terminal inactive metabolite of prednisolone, Δ(1)-cortienic acid (Δ(1)-CA), is added to HC, as Δ(1)-CA preferentially binds transcortin, liberating more HC to elicit its therapeutic effect. Skin blanching studies, which are used to evaluate the potency of topical steroids, were employed to assess the ability of Δ(1)-CA to enhance the activity of HC. The results demonstrate that Δ(1)-CA, when applied in combination with HC, does indeed potentiate the vasoconstriction effect of topically applied HC, while having no effect alone. Thus, addition of the inert prednisolone metabolite Δ(1)-CA can increase the therapeutic effect of over-the-counter concentrations of HC when applied topically. PMID:27179671

  12. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers.

    Science.gov (United States)

    Farley, Emma K; Olson, Katrina M; Zhang, Wei; Rokhsar, Daniel S; Levine, Michael S

    2016-06-01

    Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.

  13. 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 aci...

  14. Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

    Directory of Open Access Journals (Sweden)

    Stella E Autenrieth

    2012-02-01

    Full Text Available Dendritic cells (DCs as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye. We used CD11c-diphtheria toxin (DT mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

  15. Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

    Science.gov (United States)

    Shpigel, Etai; Roiz, Levava; Goren, Raphael; Shoseyov, Oded

    1998-01-01

    Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. PMID:9701575

  16. A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system.

    Science.gov (United States)

    Lewis, Melanie J; Meehan, Mary; Owen, Peter; Woof, Jenny M

    2008-06-20

    The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human). PMID:18411272

  17. Solid-State NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization

    International Nuclear Information System (INIS)

    Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool. (authors)

  18. Fructose-enhanced reduction of bacterial growth on nanorough surfaces

    Directory of Open Access Journals (Sweden)

    Durmus NG

    2012-02-01

    Full Text Available Naside Gozde Durmus1, Erik N Taylor1, Fatih Inci3,4, Kim M Kummer1, Keiko M Tarquinio5, Thomas J Webster1,21School of Engineering, Brown University, Providence, RI, USA; 2Department of Orthopedics, Brown University, Providence, RI, USA; 3Bio-Acoustic-MEMS in Medicine (BAMM Laboratory, Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard-MIT Health Sciences and Technology, Harvard Medical School, MA, USA; 4Istanbul Technical University, Molecular Biology-Genetics and Biotechnology Program, Mobgam, Maslak, Istanbul, Turkey; 5Division of Pediatric Critical Care Medicine, Rhode Island Hospital, Providence, RI, USAAbstract: Patients on mechanical ventilators for extended periods of time often face the risk of developing ventilator-associated pneumonia. During the ventilation process, patients incapable of breathing are intubated with polyvinyl chloride (PVC endotracheal tubes (ETTs. PVC ETTs provide surfaces where bacteria can attach and proliferate from the contaminated oropharyngeal space to the sterile bronchoalveolar area. To overcome this problem, ETTs can be coated with antimicrobial agents. However, such coatings may easily delaminate during use. Recently, it has been shown that changes in material topography at the nanometer level can provide antibacterial properties. In addition, some metabolites, such as fructose, have been found to increase the efficiency of antibiotics used to treat Staphylococcus aureus (S. aureus infections. In this study, we combined the antibacterial effect of nanorough ETT topographies with sugar metabolites to decrease bacterial growth and biofilm formation on ETTs. We present for the first time that the presence of fructose on the nanorough surfaces decreases the number of planktonic S. aureus bacteria in the solution and biofilm formation on the surface after 24 hours. We thus envision that this method has the potential to impact the future of surface engineering of

  19. Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor

    DEFF Research Database (Denmark)

    Gerlach, Lars Ole; Jakobsen, Janus S; Jensen, Kasper P;

    2003-01-01

    +), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion...... demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding...... of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced...

  20. Enhancement of flagellated bacterial motility in polymer solutions

    Science.gov (United States)

    Zhang, Wenyu; Sha, Sha; Pelcovits, Robert; Tang, Jay

    2015-11-01

    Measurements of the swimming speed of many species of flagellated bacteria in polymer solutions have shown that with the addition of high molecular weight polymers, the speed initially increases as a function of the kinematic viscosity. It peaks at around 1.5-2 cP with typically 10-30% higher values than in cell media without added polymers (~ 1 cP). Past the peak, the average speed gradually decreases as the solution becomes more viscous. Swimming motility persists until solution viscosity reaches 5-10 cP. Models have been proposed to account for this behavior, and the magnitude of the peak becomes a crucial test of theoretical predictions. The status of the field is complicated in light of a recent report (Martinez et al., PNAS, 2014), stressing that low-molecular weight impurities account for the peaked speed-viscosity curves in some cases. We measured the swimming speed of a uni-flagellated bacterium, caulobacter crescentus, in solutions of a number of polymers of several different sizes. Our findings confirm the peaked speed-viscosity curve, only as the molecular weight of the flexible polymers used surpassed ~ 50,000 da. The threshold molecular weight required to augment swimming speed varies somewhat with the polymer species, but it generally corresponds to radius of gyration over tens of nanometers. This general feature is consistent with the model of Powers et al. (Physics of Fluid, 2009), predicting that nonlinear viscoelasticity of the fluid enhances swimming motility. Work Supported by the NSF Fluid Physics Program (Award number CBET 1438033).

  1. Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

    Science.gov (United States)

    Hayward, Richard D; Cain, Robert J; McGhie, Emma J; Phillips, Neil; Garner, Matthew J; Koronakis, Vassilis

    2005-05-01

    A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

  2. Characterization of the hydrophobic substrate-binding site of the bacterial beta class glutathione transferase from Proteus mirabilis.

    Science.gov (United States)

    Federici, Luca; Masulli, Michele; Di Ilio, Carmine; Allocati, Nerino

    2010-09-01

    Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes.

  3. Enhancing biocompatibility of some cation selective electrodes using heparin modified bacterial cellulose.

    Science.gov (United States)

    Badr, Ibrahim H A; Abdel-Sattar, R; Keshk, Sherif M A S

    2015-12-10

    Bacterial cellulose (BC) and heparin-modified bacterial cellulose (HBC) were utilized to enhance the biocompatibility of highly thrombogenic PVC-based potassium and calcium membrane electrodes. Three types of membrane electrodes were prepared: (1) conventional PVC electrode (control), (2) PVC-based electrode sandwiched with bacterial cellulose membrane (BC-PVC), and (3) PVC-based electrode sandwiched with heparin-modified bacterial cellulose membrane (HBC-PVC). The potentiometric response characteristics of the modified potassium and calcium membrane electrodes (BC-PVC and HBC-PVC) were compared with those of the control PVC-based potassium and calcium selective electrode, respectively. Response characteristics of the modified membrane electrodes were comparable to the control PVC membrane electrode. The platelet adhesion investigations indicated that (BC) and (HBC) layers are less thrombogenic compared to PVC. Therefore, use of BC or HBC would enable the enhancement of the biocompatibility of PVC-based membrane electrodes for potassium and calcium while practically maintaining the overall electrochemical performance of the PVC sensing film. PMID:26428173

  4. Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA%新型超耐热菌的单链DNA结合蛋白表达和纯化及其增强DNA、cDNA的合成

    Institute of Scientific and Technical Information of China (English)

    贾晓伟; 张国辉; 史海燕

    2012-01-01

    目的 表达和纯化一种新的来自于超耐热菌(thermococcus kodakarensis) KOD1的单链DNA结合蛋白(缩写为kod-ssb),并探讨其对DNA、cDNA合成的影响.方法 采用Transrtta(DE3)表达kod-ssb,用镍柱亲和层析纯化,经SDS-PAGE分析检测表达纯化后的kod-ssb.使用PCR及qRT/qPCR检测kod-ssb对DNA、cDNA合成的影响.结果 将质粒pET11a-kod转化Transetta(DE3)中,得到重组菌株Transetta(pET11a-kod),经IPTG诱导.由SDS-PAGE分析可见表达的约40×103的特异条带;使用人类β球蛋白基因作为模板分别扩增5 kbp,9 kbp和13 kbp目的片段,结果加了kod-ssb的PCR目的片段比没加的产量高很多,而且kod-ssb显著降低了PCR反应的非特异性扩增.以流感细胞培养上清抽提的RNA为模板,实施RT-/qPCR反应.结果加kod的平均Ct值是19.42,而没加的为22.15.结论 kod-ssb在未来将被用于增强DNA和cDNA的合成.%Objective Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1,abbreviated kod-ssb.And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.Methods We express kod-ssb with the Transrtta (DE3),and kod-ssb was purified by affinity chromatography on a Ni2 + Sepharose column,detected by SDS-PAGE.To evaluate the effect of kod-ssb on PCR-based DNA amplification,the human beta globin gene was used as template to amplify a 5-kb,9-kb and 13-kb.And to detect the effect of kod-ssb on reverse transcription,we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.Results The plasmid pET11 a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta(pET11a-kod)was obtained.The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod)was induced by IPTG.The specific protein was detected by SDS-PAGE.To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products,5-,9-,and 13-kb human beta

  5. Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkholderia ambifaria.

    Science.gov (United States)

    Richichi, Barbara; Imberty, Anne; Gillon, Emilie; Bosco, Rosa; Sutkeviciute, Ieva; Fieschi, Franck; Nativi, Cristina

    2013-06-28

    Burkholderia ambifaria is a bacterium member of the Burkholderia cepacia complex (BCC), a closely related group of Gram-negative bacteria responsible for "cepacia syndrome" in immunocompromised patients. B. ambifaria produces BambL, a fucose-binding lectin that displays fine specificity to human fucosylated epitopes. Here, we report the first example of a synthetic ligand able to selectively bind, in the micromolar range, the pathogen-lectin BambL. The synthetic routes for the preparation of the α conformationally constrained fucoside are described, focusing on a totally diastereoselective inverse electron demand [4 + 2] Diels-Alder reaction. Isothermal titration calorimetry (ITC) demonstrated that this compound binds to the pathogen-associated lectin BambL with an affinity comparable to that of natural fucose-containing oligosaccharides. No binding was observed by LecB, a fucose-binding lectin from Pseudomonas aeruginosa, and the differences in affinity between the two lectins could be rationalized by modeling. Furthermore, SPR analyses showed that this fucomimetic does not bind to the human fucose-binding lectin DC-SIGN, thus supporting the selective binding profile towards B. ambifaria lectin.

  6. Perinatal Exposure to Environmental Tobacco Smoke (ETS Enhances Susceptibility to Viral and Secondary Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Jocelyn A. Claude

    2012-10-01

    Full Text Available Studies suggest childhood exposure to environmental tobacco smoke (ETS leads to increased incidence of infections of the lower respiratory tract. The objective of this study was to determine whether perinatal exposure to ETS increases the incidence, morbidity and severity of respiratory influenza infection and whether a secondary bacterial challenge at the peak of a pre-existing viral infection creates an enhanced host-pathogen susceptibility to an opportunistic infection. Timed-pregnant female Balb/c mice were exposed to either ETS for 6 h/day, 7 d/week beginning on gestation day 14 and continuing with the neonates to 6 weeks of age. Control animals were exposed to filtered air (FA. At the end of exposure, mice were intranasally inoculated with a murine-adapted influenza A. One week later, an intranasal inoculation of S. aureus bacteria was administered. The respective treatment groups were: bacteria only, virus only or virus+bacteria for both FA and ETS-exposed animals for a total of six treatment groups. Animal behavior and body weights were documented daily following infection. Mice were necropsied 1-day post-bacterial infection. Bronchoalveolar lavage fluid (BALF cell analysis demonstrated perinatal exposure to ETS, compared to FA, leads to delayed but enhanced clinical symptoms and enhanced total cell influx into the lungs associated with viral infection followed by bacterial challenge. Viral infection significantly increases the number of neutrophils entering the lungs following bacterial challenge with either FA or ETS exposure, while the influx of lymphocytes and monocytes is significantly enhanced only by perinatal ETS exposure. There is a significant increase in peribronchiolar inflammation following viral infection in pups exposed to ETS compared with pups exposed to FA, but no change is noted in the degree of lung injury between FA and ETS-exposed animals following bacterial challenge. The data suggests perinatal exposure to ETS

  7. Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

    Directory of Open Access Journals (Sweden)

    Li-Ping Bai

    Full Text Available With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18 of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC (14 as well as natural epigallocatechin (EGC, 6. The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

  8. Enhanced production of bacterial cellulose by using Gluconacetobacter hansenii NCIM 2529 strain under shaking conditions.

    Science.gov (United States)

    Mohite, Bhavna V; Salunke, Bipinchandra K; Patil, Satish V

    2013-03-01

    Bacterial cellulose (BC), a biopolymer, due to its unique properties is valuable for production of vital products in food, textile, medicine, and agriculture. In the present study, the optimal fermentation conditions for enhanced BC production by Gluconacetobacter hansenii NCIM 2529 were investigated under shaking conditions. The investigation on media components and culture parameters revealed that 2 % (w/v) sucrose as carbon source, 0.5 % (w/v) potassium nitrate as nitrogen source, 0.4 % (w/v) disodium phosphate as phosphate source, 0.04 % (w/v) magnesium sulfate, and 0.8 % (w/v) calcium chloride as trace elements, pH5.0, temperature 25 °C, and agitation speed 170 rpm with 6 days of fermentation period are optimal for maximum BC production. Production of BC using optimized media components and culture parameters was 1.66 times higher (5.0 g/l) than initial non optimized media (3.0 g/l). Fourier transform infrared spectroscopy spectrum and comparison with the available literature suggests that the produced component by G. hansenii in the present study is pure bacterial cellulose. The specific action of cellulase out of the investigated hydrolytic enzymes (cellulase, amylase, and protease) further confirmed purity of the produced BC. These findings give insight into conditions necessary for enhanced production of bacterial cellulose, which can be used for a variety of applications.

  9. Diamagnetic levitation enhances growth of liquid bacterial cultures by increasing oxygen availability.

    Science.gov (United States)

    Dijkstra, Camelia E; Larkin, Oliver J; Anthony, Paul; Davey, Michael R; Eaves, Laurence; Rees, Catherine E D; Hill, Richard J A

    2011-03-01

    Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to reproduce aspects of weightlessness, on the Earth. We used a superconducting magnet to levitate growing bacterial cultures for up to 18 h, to determine the effect of diamagnetic levitation on all phases of the bacterial growth cycle. We find that diamagnetic levitation increases the rate of population growth in a liquid culture and reduces the sedimentation rate of the cells. Further experiments and microarray gene analysis show that the increase in growth rate is owing to enhanced oxygen availability. We also demonstrate that the magnetic field that levitates the cells also induces convective stirring in the liquid. We present a simple theoretical model, showing how the paramagnetic force on dissolved oxygen can cause convection during the aerobic phases of bacterial growth. We propose that this convection enhances oxygen availability by transporting oxygen around the liquid culture. Since this process results from the strong magnetic field, it is not present in other weightless environments, e.g. in Earth orbit. Hence, these results are of significance and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena. PMID:20667843

  10. Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

    OpenAIRE

    Tomme, P; Driver, D P; Amandoron, E A; Miller, R. C.; Antony, R.; Warren, J.; Kilburn, D G

    1995-01-01

    A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex,...

  11. Investigation of spore forming bacterial flooding for enhanced oil recovery in a North Sea chalk Reservoir

    DEFF Research Database (Denmark)

    Halim, Amalia Yunita; Nielsen, Sidsel Marie; Eliasson Lantz, Anna;

    2015-01-01

    in higher oil production from the heterogeneous chalk rock. In all cases, an incubation period ('shut-in') after the bacterial and/or nutrient injection was needed to give sufficient time for the bacteria to grow inside the core and to produce more oil. Our findings show potential application of bacteria......Little has been done to study microbial enhanced oil recovery (MEOR) in chalk reservoirs. The present study focuses on core flooding experiments designed to see microbial plugging and its effect on oil recovery. A pressure tapped core holder was used for this purpose. A spore forming bacteria...... Bacillus licheniformis 421 was used as it was shown to be a good candidate in a previous study. Bacterial spore can penetrate deeper into the chalk rock, squeezing through the pore throats. Our results showed that injection of B. licheniformis 421 as a tertiary oil recovery method, in the residual oil...

  12. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B;

    2006-01-01

    by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  13. Thin stillage supplementation greatly enhances bacterial cellulose production by Gluconacetobacter xylinus.

    Science.gov (United States)

    Wu, Jyh-Ming; Liu, Ren-Han

    2012-09-01

    Thin stillage (TS), a wastewater from rice wine distillery can well sustain the growth of Gluconacetobacter xylinus for production of bacterial cellulose (BC). When used as a supplement to the traditional BC production medium (Hestrin and Schramm medium), the enhancement of BC production increased with the amount of TS supplemented in a static culture of G. xylinus. When TS was employed to replace distilled water for preparing HS medium (100%TS-HS medium), the BC production in this 100%TS-HS medium was enhanced 2.5-fold to a concentration of 10.38 g/l with sugar to BC conversion yield of 57% after 7 days cultivation. The cost-free TS as a supplement in BC production medium not only can greatly enhance the BC production, but also can effectively dispose the nuisance wastewater of rice wine distillery.

  14. Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

    DEFF Research Database (Denmark)

    Hwang, C S; Mandrup, S; MacDougald, O A;

    1996-01-01

    Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob......, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter....

  15. Hacking RNA: Hakai promotes tumorigenesis by enhancing the RNA-binding function of PSF.

    Science.gov (United States)

    Figueroa, Angélica; Fujita, Yasuyuki; Gorospe, Myriam

    2009-11-15

    Hakai, an E3 ubiquitin ligase for the E-cadherin complex, plays a crucial role in lowering cell-cell contacts in epithelial cells, a hallmark feature of tumor progression. Recently, Hakai was also found to interact with PSF (PTB-associated splicing factor). While PSF can function as a DNA-binding protein with a tumor suppressive function, its association with Hakai promotes PSF's RNA-binding ability and post-transcriptional influence on target mRNAs. Hakai overexpression enhanced the binding of PSF to mRNAs encoding cancer-related proteins, while knockdown of Hakai reduced the RNA-binding ability of PSF. Furthermore, the knockdown of PSF suppressed Hakai-induced cell proliferation. Thus, Hakai can affect the oncogenic phenotype both by altering E-cadherin-based intercellular adhesions and by increasing PSF's ability to bind RNAs that promote cancer-related gene expression.

  16. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation.

    Science.gov (United States)

    Jeon, Jongho; Lee, Kyung Hyun; Rao, Jianghong

    2012-10-14

    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

  17. Bacterial-binding chitosan microspheres for gastric infection treatment and prevention.

    Science.gov (United States)

    Gonçalves, Inês C; Magalhães, Ana; Fernandes, Mariana; Rodrigues, Inês V; Reis, Celso A; Martins, M Cristina L

    2013-12-01

    Helicobacter pylori (H. pylori) colonizes the gastric mucosa of over 50% of the world population, causing several pathologies, such as gastric ulcers and gastric cancer. Since current antibiotic treatments are inefficient in 20% of cases alternative therapies are needed. This work reports the ability of chitosan microspheres to adhere to H. pylori and prevent/remove H. pylori colonization. Adhesion of H. pylori strains with different functional adhesins (BabA and/or SabA) to chitosan microspheres (diameter 167 ± 27 μm) occurs at both pH 2.6 and 6.0, but is higher at pH 6.0. Bacterial adhesion to a gastric cell line expressing sialylated carbohydrates (SabA receptors) was performed at the same pH values using H. pylori strains with and without SabA. At both pH values addition of microspheres to gastric cells before and after pre-incubation with H. pylori decreased bacterial adhesion to cells. Furthermore, the chitosan microspheres were non-cytotoxic. These findings reveal the potential of chitosan microspheres as an alternative or complementary treatment for H. pylori gastric eradication or prevention of H. pylori colonization.

  18. Bacterial stress enrichment enhances anaerobic hydrogen production in cattle manure sludge.

    Science.gov (United States)

    Cheong, Dae-Yeol; Hansen, Conly L

    2006-10-01

    Methodology was evaluated to selectively enrich hydrogen-producing species present in biological sludge produced during organic wastewater treatment. The influence of bacterial stress enrichment on anaerobic hydrogen-producing microorganisms was investigated in batch tests using serum bottles. Enrichment conditions investigated included application of acute physical and chemical stresses: wet heat, dry heat and desiccation, use of a methanogen inhibitor, freezing and thawing, and chemical acidification with and without preacidification of the sludge at pH 3. For each enrichment sample, cultivation pH value was set at an initial value of 7. After application of selective enrichment (by bacterial stress), hydrogen production was significantly higher than that of untreated original sludge. Hydrogen production from the inocula with bacterial stress enrichment was 1.9-9.8 times greater when compared with control sludge. Chemical acidification using perchloric acid showed the best hydrogen production potential, irrespective of preacidification. Enhancement is due to the selective capture of hydrogen-producing sporeformers, which induces altered anaerobic fermentative metabolism. PMID:16525779

  19. Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco.

    Science.gov (United States)

    Vogel-Adghough, Drissia; Stahl, Elia; Návarová, Hana; Zeier, Juergen

    2013-11-01

    Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.

  20. DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

    Directory of Open Access Journals (Sweden)

    Didych Dmitry A

    2012-04-01

    Full Text Available Abstract Background Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. Results Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. Conclusions We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

  1. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    Science.gov (United States)

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  2. Raman and surface-enhanced Raman scattering (SERS) studies of the thrombin-binding aptamer.

    Science.gov (United States)

    Wu, Tsai-Chin; Vasudev, Milana; Dutta, Mitra; Stroscio, Michael A

    2013-06-01

    Surface-enhanced Raman scattering is used to study the Raman spectra and peak shifts the thrombin-binding aptamer (TBA) on substrates having two different geometries; one with a single stranded sequence and one with double stranded sequence. The Raman signals of the deoxyribonucleic acids on both substrates are enhanced and specific peaks of bases are identified. These results are highly reproducible and have promising applications in low cost nucleic acid detection.

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

    Science.gov (United States)

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

    2013-10-01

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

  4. Nonlinearity arising from noncooperative transcription factor binding enhances negative feedback and promotes genetic oscillations

    CERN Document Server

    Lengyel, Iván M; Oates, Andrew C; Morelli, Luis G

    2015-01-01

    We study the effects of multiple binding sites in the promoter of a genetic oscillator. We evaluate the regulatory function of a promoter with multiple binding sites in the absence of cooperative binding, and consider different hypotheses for how the number of bound repressors affects transcription rate. Effective Hill exponents of the resulting regulatory functions reveal an increase in the nonlinearity of the feedback with the number of binding sites. We identify optimal configurations that maximize the nonlinearity of the feedback. We use a generic model of a biochemical oscillator to show that this increased nonlinearity is reflected in enhanced oscillations, with larger amplitudes over wider oscillatory ranges. Although the study is motivated by genetic oscillations in the zebrafish segmentation clock, our findings may reveal a general principle for gene regulation.

  5. The chicken CCAAT/Enhancer Binding Protein alpha gene. Cloning, characterisation and tissue distribution

    NARCIS (Netherlands)

    Calkhoven, CF; Gringhuis, SI; Ab, G

    1997-01-01

    We present the cloning and sequencing of the gene encoding the chicken CCAAT/Enhancer Binding Protein alpha (cC/EBP alpha). The coding region and 1.5 kb of 5' flanking DNA form a CpG island. Comparison of the chicken C/EBP alpha sequence to the homologous proteins of other species reveals several ev

  6. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake.

    Science.gov (United States)

    Iyer, Abhishek; Van Lysebetten, Dorien; Ruiz García, Yara; Louage, Benoit; De Geest, Bruno G; Madder, Annemieke

    2015-04-01

    The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.

  7. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  8. Secondary Structure Preferences of Mn2+ Binding Sites in Bacterial Proteins

    Directory of Open Access Journals (Sweden)

    Tatyana Aleksandrovna Khrustaleva

    2014-01-01

    Full Text Available 3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used. Major Mn2+ binders are aspartic acid (6.82% of Asp residues, histidine (14.76% of His residues, and glutamic acid (3.51% of Glu residues. We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88% does not depend on genomic GC-content.

  9. Uncovering the Transmembrane Metal Binding Site of the Novel Bacterial Major Facilitator Superfamily-Type Copper Importer CcoA

    Directory of Open Access Journals (Sweden)

    Bahia Khalfaoui-Hassani

    2016-01-01

    Full Text Available Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox. Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive 64Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter.

  10. Enhancement of binding kinetics on affinity substrates by laser point heating induced transport.

    Science.gov (United States)

    Wang, Bu; Cheng, Xuanhong

    2016-03-01

    Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used the COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates is quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces the reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predictable binding patterns. This optothermal strategy holds promise to improve the time-response and sensitivity of biosensors and microarrays. PMID:26898559

  11. Magnesium aminoclay enhances lipid production of mixotrophic Chlorella sp. KR-1 while reducing bacterial populations.

    Science.gov (United States)

    Kim, Bohwa; Praveenkumar, Ramasamy; Lee, Jiye; Nam, Bora; Kim, Dong-Myung; Lee, Kyubock; Lee, Young-Chul; Oh, You-Kwan

    2016-11-01

    Improving lipid productivity and preventing overgrowth of contaminating bacteria are critical issues relevant to the commercialization of the mixotrophic microalgae cultivation process. In this paper, we report the use of magnesium aminoclay (MgAC) nanoparticles for enhanced lipid production from oleaginous Chlorella sp. KR-1 with simultaneous control of KR-1-associated bacterial growth in mixotrophic cultures with glucose as the model substrate. Addition of 0.01-0.1g/L MgAC promoted microalgal biomass production better than the MgAC-less control, via differential biocidal effects on microalgal and bacterial cells (the latter being more sensitive to MgAC's bio-toxicity than the former). The inhibition effect of MgAC on co-existing bacteria was, as based on density-gradient-gel-electrophoresis (DGGE) analysis, largely dosage-dependent and species-specific. MgAC also, by inducing an oxidative stress environment, increased both the cell size and lipid content of KR-1, resulting in a considerable, ∼25% improvement of mixotrophic algal lipid productivity (to ∼410mgFAME/L/d) compared with the untreated control. PMID:27543952

  12. Magnesium aminoclay enhances lipid production of mixotrophic Chlorella sp. KR-1 while reducing bacterial populations.

    Science.gov (United States)

    Kim, Bohwa; Praveenkumar, Ramasamy; Lee, Jiye; Nam, Bora; Kim, Dong-Myung; Lee, Kyubock; Lee, Young-Chul; Oh, You-Kwan

    2016-11-01

    Improving lipid productivity and preventing overgrowth of contaminating bacteria are critical issues relevant to the commercialization of the mixotrophic microalgae cultivation process. In this paper, we report the use of magnesium aminoclay (MgAC) nanoparticles for enhanced lipid production from oleaginous Chlorella sp. KR-1 with simultaneous control of KR-1-associated bacterial growth in mixotrophic cultures with glucose as the model substrate. Addition of 0.01-0.1g/L MgAC promoted microalgal biomass production better than the MgAC-less control, via differential biocidal effects on microalgal and bacterial cells (the latter being more sensitive to MgAC's bio-toxicity than the former). The inhibition effect of MgAC on co-existing bacteria was, as based on density-gradient-gel-electrophoresis (DGGE) analysis, largely dosage-dependent and species-specific. MgAC also, by inducing an oxidative stress environment, increased both the cell size and lipid content of KR-1, resulting in a considerable, ∼25% improvement of mixotrophic algal lipid productivity (to ∼410mgFAME/L/d) compared with the untreated control.

  13. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    Science.gov (United States)

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  14. Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers

    Science.gov (United States)

    Liu, Chia-Ying; Han, Yin-Yi; Shih, Po-Han; Lian, Wei-Nan; Wang, Huai-Hsien; Lin, Chi-Hung; Hsueh, Po-Ren; Wang, Juen-Kai; Wang, Yuh-Lin

    2016-01-01

    Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology. PMID:26997474

  15. Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean.

    Science.gov (United States)

    Wang, Jialin; Shine, M B; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

    2014-05-28

    Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector.

  16. The meningococcal vaccine candidate neisserial surface protein A (NspA binds to factor H and enhances meningococcal resistance to complement.

    Directory of Open Access Journals (Sweden)

    Lisa A Lewis

    Full Text Available Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH to fH-binding protein (fHbp is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a approximately 17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA, a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep I chain of lipooligosaccharide (LOS, or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6-7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

  17. Efficacy of coating activated carbon with milk proteins to prevent binding of bacterial cells from foods for PCR detection.

    Science.gov (United States)

    Opet, Nathan J; Levin, Robert E

    2013-08-01

    Foods contaminated with pathogens are common sources of illness. Currently, the most common and sensitive rapid detection method involves the PCR. However, food matrices are complex and contain inhibitors that limit the sensitivity of the PCR. The use of coated activated carbon can effectively facilitate the removal of PCR inhibitors without binding targeted bacterial cells from food samples. With the use of activated carbon coated with milk proteins, a cell recovery at pH 7.0 of 95.7%±2.0% was obtained, compared to control uncoated activated carbon, which yielded a cell recovery of only 1.1%±0.8%. In addition, the milk protein coated activated carbon was able to absorb similar amounts of soluble compounds as uncoated activated carbon, with the exception of bovine hemoglobin. This suggests that the use of milk proteins to coat activated carbon may therefore serve as a suitable replacement for bentonite in the coating of activated carbon, which has previously been used for the removal of PCR inhibitors from food.

  18. Enhancement effect of terbium complex luminescence by binding to silver nanoparticles in the solution

    Institute of Scientific and Technical Information of China (English)

    WANG Yue-hui; ZHOU Ji; ZONG Rui-long; SHI Shi-kao; WANG Ting; LI Bo

    2006-01-01

    The luminescent properties of terbium complex (terbium citrate) by binding to silver nanopartilces in the solution have been reported in this paper.The enhanced luminescence of the complex containing silver nanoparticles was observed at a limited particle concentration region.The nanoparticle concentration dependence of the luminescent intensity was regarded as the result of a delicate balance between an enhancing and a quenching effect of the silver nanoparticles.Furthermore,silver nanoparticles also affected the asymmetric ratio (AS) value of terbium luminescence.We discuss the luminescent properties of the terbium complex in terms of the local electromagnetic field,refractive index,and the ligand field around terbium ion.

  19. Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection.

    Science.gov (United States)

    Sistrunk, Jeticia R; Nickerson, Kourtney P; Chanin, Rachael B; Rasko, David A; Faherty, Christina S

    2016-10-01

    Bacterial pathogens have coevolved with humans in order to efficiently infect, replicate within, and be transmitted to new hosts to ensure survival and a continual infection cycle. For enteric pathogens, the ability to adapt to numerous host factors under the harsh conditions of the gastrointestinal tract is critical for establishing infection. One such host factor readily encountered by enteric bacteria is bile, an innately antimicrobial detergent-like compound essential for digestion and nutrient absorption. Not only have enteric pathogens evolved to resist the bactericidal conditions of bile, but these bacteria also utilize bile as a signal to enhance virulence regulation for efficient infection. This review provides a comprehensive and up-to-date analysis of bile-related research with enteric pathogens. From common responses to the unique expression of specific virulence factors, each pathogen has overcome significant challenges to establish infection in the gastrointestinal tract. Utilization of bile as a signal to modulate virulence factor expression has led to important insights for our understanding of virulence mechanisms for many pathogens. Further research on enteric pathogens exposed to this in vivo signal will benefit therapeutic and vaccine development and ultimately enhance our success at combating such elite pathogens. PMID:27464994

  20. EXPRESSION OF BACTERIAL PROTEIN-A IN TOBACCO LEADS TO ENHANCED RESISTANCE TO STRESS CONDITIONS

    Directory of Open Access Journals (Sweden)

    Chaitali Roy

    2014-08-01

    Full Text Available Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms because it can be easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. As bacterial enzymes can be synthesized in tobacco, here we explore the possibility of in planta expression of staphylococcal protein-A(PA which is an antibody, an important group among biopharmaceuticals. In our study we have shown that the tobacco plants harboring PA gene could combat the crown gall infection and also effective in resisting abiotic stress conditions. Transgenic plants when subjected to interact with wild variety of Agrobacterium shows its enhanced capability to resist the gall formation. And when transgenic tobacco plants were grown in presence of 200mM NaCl and/or MG(Methylglyoxal solution, shows their increased tolerance towards salinity stress and high MG stress. So far transgenic tobacco plants are concerned, improvements in the expression of recombinant proteins and their recovery from tobacco may also enhance production and commercial use of this protein.

  1. Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis

    Directory of Open Access Journals (Sweden)

    Demirci Ali

    2009-07-01

    Full Text Available Abstract Bacterial cellulose has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated foods. It has also been used in the paper manufacturing industry to enhance paper strength, the electronics industry in acoustic diaphragms for audio speakers, the pharmaceutical industry as filtration membranes, and in the medical field as wound dressing and artificial skin material. In this study, different types of plastic composite support (PCS were implemented separately within a fermentation medium in order to enhance bacterial cellulose (BC production by Acetobacter xylinum. The optimal composition of nutritious compounds in PCS was chosen based on the amount of BC produced. The selected PCS was implemented within a bioreactor to examine the effects on BC production in a batch fermentation. The produced BC was analyzed using X-ray diffraction (XRD, field emission scanning electron microscopy (FESEM, thermogravimetric analysis (TGA, and dynamic mechanical analysis (DMA. Among thirteen types of PCS, the type SFYR+ was selected as solid support for BC production by A. xylinum in a batch biofilm reactor due to its high nitrogen content, moderate nitrogen leaching rate, and sufficient biomass attached on PCS. The PCS biofilm reactor yielded BC production (7.05 g/L that was 2.5-fold greater than the control (2.82 g/L. The XRD results indicated that the PCS-grown BC exhibited higher crystallinity (93% and similar crystal size (5.2 nm to the control. FESEM results showed the attachment of A. xylinum on PCS, producing an interweaving BC product. TGA results demonstrated that PCS-grown BC had about 95% water retention ability, which was lower than BC produced within suspended-cell reactor. PCS-grown BC also exhibited higher Tmax compared to the control. Finally, DMA results showed that BC from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young's modulus when compared to

  2. Target-specific capture enhances sensitivity of electrochemical detection of bacterial pathogens.

    Science.gov (United States)

    Patel, Mayank; Gonzalez, Rodrigo; Halford, Colin; Lewinski, Michael A; Landaw, Elliot M; Churchill, Bernard M; Haake, David A

    2011-12-01

    We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay.

  3. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    Science.gov (United States)

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

  4. RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

    Directory of Open Access Journals (Sweden)

    Charles R. Cantor

    2011-03-01

    Full Text Available Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.

  5. Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.

    Science.gov (United States)

    Ribitsch, Doris; Yebra, Antonio Orcal; Zitzenbacher, Sabine; Wu, Jing; Nowitsch, Susanne; Steinkellner, Georg; Greimel, Katrin; Doliska, Ales; Oberdorfer, Gustav; Gruber, Christian C; Gruber, Karl; Schwab, Helmut; Stana-Kleinschek, Karin; Acero, Enrique Herrero; Guebitz, Georg M

    2013-06-10

    A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ μM (native enzyme) to 0.21 and 0.33 s(-1)/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

  6. Impact of bacterial vaginosis, as assessed by nugent criteria and hormonal status on glycosidases and lectin binding in cervicovaginal lavage samples.

    Directory of Open Access Journals (Sweden)

    Bernard J Moncla

    Full Text Available The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA. Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively. The amount of sialic acid (Sambucus nigra, P = 0.003 and high mannose (griffithsin, P<0.001 were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02 when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.

  7. DNA Bending is Induced in an Enhancer by the DNA-Binding Domain of the Bovine Papillomavirus E2 Protein

    Science.gov (United States)

    Moskaluk, Christopher; Bastia, Deepak

    1988-03-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I ``footprinting'' on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.

  8. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

    OpenAIRE

    Jonathan Göke; Marc Jung; Sarah Behrens; Lukas Chavez; Sean O'Keeffe; Bernd Timmermann; Hans Lehrach; James Adjaye; Martin Vingron

    2011-01-01

    Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-lo...

  9. A comparative study of the bacterial community in denitrifying and traditional enhanced biological phosphorus removal processes.

    Science.gov (United States)

    Lv, Xiao-Mei; Shao, Ming-Fei; Li, Chao-Lin; Li, Ji; Gao, Xin-Lei; Sun, Fei-Yun

    2014-09-17

    Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. Two lab-scale sequencing batch reactors (SBRs) were operated in alternating anaerobic-anoxic (A-A) or anaerobic-oxic (A-O) conditions to achieve denitrifying enhanced biological phosphate removal (EBPR) and traditional EBPR. No significant differences were observed in phosphorus removal efficiencies between A-A SBR and A-O SBR, with phosphorus removal rates being 87.9% and 89.0% respectively. The community structures in denitrifying and traditional EBPR processes were evaluated by high-throughput sequencing of the PCR-amplified partial 16S rRNA genes from each sludge. The results obtained showed that the bacterial community was more diverse in A-O sludge than in A-A sludge. Taxonomy and β-diversity analyses indicated that a significant shift occurred in the dominant microbial community in A-A sludge compared with the seed sludge during the whole acclimation phase, while a slight fluctuation was observed in the abundance of the major taxonomies in A-O sludge. One Dechloromonas-related OTU outside the 4 known Candidatus "Accumulibacter" clades was detected as the main OTU in A-A sludge at the stationary operation, while Candidatus "Accumulibacter" dominated in A-O sludge. PMID:24964811

  10. MspA Porin-Gold Nanoparticle Assemblies: Enhanced Binding through a Controlled Cysteine Mutation.

    Science.gov (United States)

    Dani, Raj Kumar; Kang, Myungshim; Kalita, Mausam; Smith, Paul E; Bossmann, Stefan H; Chikan, Viktor

    2008-04-01

    In this study, the interactions of two gold nanoparticles of different sizes (average diameters of 3.7 +/- 2.6 and 17 +/- 3 nm) with octameric mycobacterial porin A from Mycobacterium smegmatis (MspA) and a mutant of MspA featuring a cysteine mutation in position 126 (Q126C) are investigated. From the observation of enhanced photoluminescence quenching, it is inferred that the presence of eight cysteines in the MspA Q126C mutant significantly enhances the binding of selected small gold nanoparticles within the inner pore of MspA. The large gold nanoparticle/porin complex shows photoluminescence enhancement, which is expected since the larger nanoparticles cannot dock within the homopore of MspA due to size exclusion. In addition to the fluorescence experiments, observation of energy transfer from the small gold nanoparticles to the MspA shows the close proximity of the small gold nanoparticles with the porin. Interestingly, the energy transfer of the large nanoparticle/MspA complex is completely missing. From high-performance liquid chromatography data, the estimated binding constants for small Au@MspA, large Au@MspA, small Au@MspAcys, and large Au@MspAcys are 1.3 x 10 (9), 2.22 x 10 (10), > 10 (12) (irreversible), and 1.7 x 10 (10), respectively. PMID:18318505

  11. Contrast-enhanced MR imaging in bacterial meningitis in children. Temporal profile and correlation with the prognosis

    International Nuclear Information System (INIS)

    Treatment periods for bacterial meningitis are often very long, and often this prolonged treatment is based on the judgment of its effectiveness by the degree of enhancement on brain magnetic resonance imaging (MRI). In this study, we analyzed the contrast MRI in the acute and recovery phases of bacterial meningitis in twelve patients, and graded the contrast level of the subdural space and subarachnoid space separately. While the contrast level of the subarachnoid space increased with time in 4 cases, that of the subdural space increased in 10 cases, and 9 of them revealed a good prognosis without continuation of the treatment. These findings indicate that increased contrast level of the subdural space is common in the recovery phase of bacterial meningitis, and that repetitive MRI investigation is not valuable to determine the duration of treatment. (author)

  12. Temperature-enhanced association of proteins due to electrostatic interaction: a coarse-grained simulation of actin-myosin binding.

    Science.gov (United States)

    Okazaki, Kei-ichi; Sato, Takato; Takano, Mitsunori

    2012-05-30

    Association of protein molecules constitutes the basis for the interaction network in a cell. Despite its fundamental importance, the thermodynamic aspect of protein-protein binding, particularly the issues relating to the entropy change upon binding, remains elusive. The binding of actin and myosin, which are vital proteins in motility, is a typical example, in which two different binding mechanisms have been argued: the binding affinity increases with increasing temperature and with decreasing salt-concentration, indicating the entropy-driven binding and the enthalpy-driven binding, respectively. How can these thermodynamically different binding mechanisms coexist? To address this question, which is of general importance in understanding protein-protein bindings, we conducted an in silico titration of the actin-myosin system by molecular dynamics simulation using a residue-level coarse-grained model, with particular focus on the role of the electrostatic interaction. We found a good agreement between in silico and in vitro experiments on the salt-concentration dependence and the temperature dependence of the binding affinity. We then figured out how the two binding mechanisms can coexist: the enthalpy (due to electrostatic interaction between actin and myosin) provides the basal binding affinity, and the entropy (due to the orientational disorder of water molecules) enhances it at higher temperatures. In addition, we analyzed the actin-myosin complex structures observed during the simulation and obtained a variety of weak-binding complex structures, among which were found an unusual binding mode suggested by an earlier experiment and precursor structures of the strong-binding complex proposed by electron microscopy. These results collectively indicate the potential capability of a residue-level coarse-grained model to simulate the association-dissociation dynamics (particularly for transient weak-bindings) exhibited by larger and more complicated systems, as in a

  13. Salt-enhanced chemical weathering of building materials and bacterial mineralization of calcium carbonate as a treatment

    Science.gov (United States)

    Schiro, M.; Ruiz-Agudo, E.; Jroundi, F.; Gonzalez-Muñoz, M. T.; Rodriguez-Navarro, C.

    2012-04-01

    Salt weathering is an important mechanism contributing to the degradation and loss of stone building materials. In addition to the physical weathering resulting from crystallization pressure, the presence of salts in solution greatly enhances the chemical weathering potential of pore waters. Flow through experiments quantify the dissolution rates of calcite and quartz grains (63-125 micrometer diameter) when subjected to 1.0 ionic strength solutions of MgSO4, MgCl, Na2SO4 or NaCl. Results indicate that the identity of the cation is the primary control over the dissolution rate of both calcite and quartz substrates, with salt-enhanced dissolution occurring most rapidly in Mg2+ bearing solutions. It has been observed that weathering rates of rocks in nature, as well as building stones, are slowed down by naturally occurring or artificially produced patinas. These tend to be bacterially produced, durable mineralized coatings that lend some degree of protection to the underlying stone surface [1]. Our research shows that bacterially produced carbonate coatings can be quite effective at reducing chemical weathering of stone by soluble salts. The calcite-producing-bacteria used in this study were isolated from stone monuments in Granada, Spain [2] and cultivated in an organic-rich culture medium on a variety of artificial and natural substrates (including limestone, marble, sandstone, quartz, calcite single crystals, glass cover-slips, and sintered porous glass). Scanning electron microscopy (FESEM) was used to image bacterial calcite growth and biofilm formation. In-situ atomic force microscopy (AFM) enabled calculation of dissolution rates of untreated and bacterially treated surfaces. 2D-XRD showed the mineralogy and crystallographic orientation of bacterial calcium carbonate. Results indicate that bacterially produced calcite crystals form a coherent, mechanically resistant surface layer in perfect crystallographic continuity with the calcite substrate (self

  14. The colitis-associated transcriptional profile of commensal Bacteroides thetaiotaomicron enhances adaptive immune responses to a bacterial antigen.

    Directory of Open Access Journals (Sweden)

    Jonathan J Hansen

    Full Text Available BACKGROUND: Inflammatory bowel diseases (IBD may be caused in part by aberrant immune responses to commensal intestinal microbes including the well-characterized anaerobic gut commensal Bacteroides thetaiotaomicron (B. theta. Healthy, germ-free HLA-B27 transgenic (Tg rats develop chronic colitis when colonized with complex gut commensal bacteria whereas non-transgenic (nTg rats remain disease-free. However, the role of B. theta in causing disease in Tg rats is unknown nor is much known about how gut microbes respond to host inflammation. METHODS: Tg and nTg rats were monoassociated with a human isolate of B. theta. Colonic inflammation was assessed by histologic scoring and tissue pro-inflammatory cytokine measurement. Whole genome transcriptional profiling of B. theta recovered from ceca was performed using custom GeneChips and data analyzed using dChip, Significance Analysis of Microarrays, and Gene Set Enrichment Analysis (GSEA software. Western Blots were used to determine adaptive immune responses to a differentially expressed B. theta gene. RESULTS: B. theta monoassociated Tg rats, but not nTg or germ-free controls, developed chronic colitis. Transcriptional profiles of cecal B. theta were significantly different in Tg vs. nTg rats. GSEA revealed that genes in KEGG canonical pathways involved in bacterial growth and metabolism were downregulated in B. theta from Tg rats with colitis though luminal bacterial concentrations were unaffected. Bacterial genes in the Gene Ontology molecular function "receptor activity", most of which encode nutrient binding proteins, were significantly upregulated in B. theta from Tg rats and include a SusC homolog that induces adaptive immune responses in Tg rats. CONCLUSIONS: B. theta induces colitis in HLA-B27 Tg rats, which is associated with regulation of bacterial genes in metabolic and nutrient binding pathways that may affect host immune responses. These studies of the host-microbial dialogue may lead to

  15. A novel human tectonin protein with multivalent beta-propeller folds interacts with ficolin and binds bacterial LPS.

    Directory of Open Access Journals (Sweden)

    Diana Hooi Ping Low

    Full Text Available BACKGROUND: Although the human genome database has been completed a decade ago, approximately 50% of the proteome remains hypothetical as their functions are unknown. The elucidation of the functions of these hypothetical proteins can lead to additional protein pathways and revelation of new cascades. However, many of these inferences are limited to proteins with substantial sequence similarity. Of particular interest here is the Tectonin domain-containing family of proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have identified hTectonin, a hypothetical protein in the human genome database, as a distant ortholog of the limulus galactose binding protein (GBP. Phylogenetic analysis revealed strong evolutionary conservation of hTectonin homologues from parasite to human. By computational analysis, we showed that both the hTectonin and GBP form beta-propeller structures with multiple Tectonin domains, each containing beta-sheets of 4 strands per beta-sheet. hTectonin is present in the human leukocyte cDNA library and immune-related cell lines. It interacts with M-ficolin, a known human complement protein whose ancient homolog, carcinolectin (CL5, is the functional protein partner of GBP during infection. Yeast 2-hybrid assay showed that only the Tectonin domains of hTectonin recognize the fibrinogen-like domain of the M-ficolin. Surface plasmon resonance analysis showed real-time interaction between the Tectonin domains 6 & 11 and bacterial LPS, indicating that despite forming 2 beta-propellers with its different Tectonin domains, the hTectonin molecule could precisely employ domains 6 & 11 to recognise bacteria. CONCLUSIONS/SIGNIFICANCE: By virtue of a recent finding of another Tectonin protein, leukolectin, in the human leukocyte, and our structure-function analysis of the hypothetical hTectonin, we propose that Tectonin domains of proteins could play a vital role in innate immune defense, and that this function has been conserved over several

  16. DNA bending is induced in an enhancer by the DNA-binding domain of the bovine papillomavirus E2 protein.

    OpenAIRE

    Moskaluk, C; Bastia, D

    1988-01-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I "footprinting" on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucle...

  17. WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly.

    Science.gov (United States)

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.

  18. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    Science.gov (United States)

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  19. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  20. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jason R Gerstner

    Full Text Available Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7 on sleep and long-term memory (LTM formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

  1. Musicians have enhanced audiovisual multisensory binding: experience-dependent effects in the double-flash illusion.

    Science.gov (United States)

    Bidelman, Gavin M

    2016-10-01

    Musical training is associated with behavioral and neurophysiological enhancements in auditory processing for both musical and nonmusical sounds (e.g., speech). Yet, whether the benefits of musicianship extend beyond enhancements to auditory-specific skills and impact multisensory (e.g., audiovisual) processing has yet to be fully validated. Here, we investigated multisensory integration of auditory and visual information in musicians and nonmusicians using a double-flash illusion, whereby the presentation of multiple auditory stimuli (beeps) concurrent with a single visual object (flash) induces an illusory perception of multiple flashes. We parametrically varied the onset asynchrony between auditory and visual events (leads and lags of ±300 ms) to quantify participants' "temporal window" of integration, i.e., stimuli in which auditory and visual cues were fused into a single percept. Results show that musically trained individuals were both faster and more accurate at processing concurrent audiovisual cues than their nonmusician peers; nonmusicians had a higher susceptibility for responding to audiovisual illusions and perceived double flashes over an extended range of onset asynchronies compared to trained musicians. Moreover, temporal window estimates indicated that musicians' windows (audiovisual binding. Collectively, findings indicate a more refined binding of auditory and visual cues in musically trained individuals. We conclude that experience-dependent plasticity of intensive musical experience extends beyond simple listening skills, improving multimodal processing and the integration of multiple sensory systems in a domain-general manner.

  2. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    Science.gov (United States)

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  3. Parameters that enhance the bacterial expression of active plant polyphenol oxidases.

    Directory of Open Access Journals (Sweden)

    Mareike E Dirks-Hofmeister

    Full Text Available Polyphenol oxidases (PPOs, EC 1.10.3.1 are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale. The system comprised the Escherichia coli Rosetta 2 (DE3 [pLysSRARE2] strain combined with the pET-22b(+-vector cultivated in auto-induction medium at a constant low temperature (26 °C. Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v(max of 0.48 ± 0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.

  4. Bacterial strains from floodplain soils perform different plant-growth promoting processes and enhance cowpea growth

    Directory of Open Access Journals (Sweden)

    Elaine Martins da Costa

    2016-08-01

    Full Text Available ABSTRACT Certain nodulating nitrogen-fixing bacteria in legumes and other nodule endophytes perform different plant-growth promoting processes. The objective of this study was to evaluate 26 bacterial strains isolated from cowpea nodules grown in floodplain soils in the Brazilian savannas, regarding performance of plant-growth promoting processes and ability to enhance cowpea growth. We also identified these strains by 16S rRNA sequencing. The following processes were evaluated: free-living biological nitrogen fixation (BNF, solubilization of calcium, aluminum and iron phosphates and production of indole-3-acetic acid (IAA. The abilities to nodulate and promote cowpea growth were evaluated in Leonard jars. Partial sequencing of the 16S rRNA gene identified 60 % of the strains as belonging to genus Paenibacillus. The following four genera were also identified: Bacillus, Bradyrhizobium, Enterobacter and Pseudomonas. None of the strains fixed N2 free-living. Among the strains, 80 % solubilized Ca phosphate and one solubilized Al phosphate and none solubilized Fe phosphate. The highest IAA concentrations (52.37, 51.52 and 51.00 μg mL−1 were obtained in the 79 medium with tryptophan by Enterobacter strains UFPI B5-7A, UFPI B5-4 and UFPI B5-6, respectively. Only eight strains nodulated cowpea, however, all increased production of total dry matter. The fact that the strains evaluated perform different biological processes to promote plant growth indicates that these strains have potential use in agricultural crops to increase production and environmental sustainability.

  5. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    Directory of Open Access Journals (Sweden)

    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  6. Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

    Science.gov (United States)

    Someya, Tatsuhiko; Baba, Seiki; Fujimoto, Mai; Kawai, Gota; Kumasaka, Takashi; Nakamura, Kouji

    2012-01-01

    Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA. PMID:22053080

  7. The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor

    NARCIS (Netherlands)

    Milon, P.; Tischenko, E.V.; Tomsic, J.; Caserta, E.; Folkers, G.E.; La Teana, A.; Rodnina, M.V.; Pon, C.L.; Boelens, R.; Gualerzi, C.O.

    2006-01-01

    Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low.

  8. A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi

    Science.gov (United States)

    Mayers, Teaghan J.; Bramucci, Anna R.; Yakimovich, Kurt M.; Case, Rebecca J.

    2016-01-01

    Emiliania huxleyi is a globally abundant microalga that plays a significant role in biogeochemical cycles. Over the next century, sea surface temperatures are predicted to increase drastically, which will likely have significant effects on the survival and ecology of E. huxleyi. In a warming ocean, this microalga may become increasingly vulnerable to pathogens, particularly those with temperature-dependent virulence. Ruegeria is a genus of Rhodobacteraceae whose population size tracks that of E. huxleyi throughout the alga’s bloom–bust lifecycle. A representative of this genus, Ruegeria sp. R11, is known to cause bleaching disease in a red macroalga at elevated temperatures. To investigate if the pathogenicity of R11 extends to microalgae, it was co-cultured with several cell types of E. huxleyi near the alga’s optimum (18°C), and at an elevated temperature (25°C) known to induce virulence in R11. The algal populations were monitored using flow cytometry and pulse-amplitude modulated fluorometry. Cultures of algae without bacteria remained healthy at 18°C, but lower cell counts in control cultures at 25°C indicated some stress at the elevated temperature. Both the C (coccolith-bearing) and S (scale-bearing swarming) cell types of E. huxleyi experienced a rapid decline resulting in apparent death when co-cultured with R11 at 25°C, but had no effect on N (naked) cell type at either temperature. R11 had no initial negative impact on C and S type E. huxleyi population size or health at 18°C, but caused death in older co-cultures. This differential effect of R11 on its host at 18 and 25°C suggest it is a temperature-enhanced opportunistic pathogen of E. huxleyi. We also detected caspase-like activity in dying C type cells co-cultured with R11, which suggests that programmed cell death plays a role in the death of E. huxleyi triggered by R11 – a mechanism induced by viruses (EhVs) and implicated in E. huxleyi bloom collapse. Given that E. huxleyi has

  9. A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi.

    Science.gov (United States)

    Mayers, Teaghan J; Bramucci, Anna R; Yakimovich, Kurt M; Case, Rebecca J

    2016-01-01

    Emiliania huxleyi is a globally abundant microalga that plays a significant role in biogeochemical cycles. Over the next century, sea surface temperatures are predicted to increase drastically, which will likely have significant effects on the survival and ecology of E. huxleyi. In a warming ocean, this microalga may become increasingly vulnerable to pathogens, particularly those with temperature-dependent virulence. Ruegeria is a genus of Rhodobacteraceae whose population size tracks that of E. huxleyi throughout the alga's bloom-bust lifecycle. A representative of this genus, Ruegeria sp. R11, is known to cause bleaching disease in a red macroalga at elevated temperatures. To investigate if the pathogenicity of R11 extends to microalgae, it was co-cultured with several cell types of E. huxleyi near the alga's optimum (18°C), and at an elevated temperature (25°C) known to induce virulence in R11. The algal populations were monitored using flow cytometry and pulse-amplitude modulated fluorometry. Cultures of algae without bacteria remained healthy at 18°C, but lower cell counts in control cultures at 25°C indicated some stress at the elevated temperature. Both the C (coccolith-bearing) and S (scale-bearing swarming) cell types of E. huxleyi experienced a rapid decline resulting in apparent death when co-cultured with R11 at 25°C, but had no effect on N (naked) cell type at either temperature. R11 had no initial negative impact on C and S type E. huxleyi population size or health at 18°C, but caused death in older co-cultures. This differential effect of R11 on its host at 18 and 25°C suggest it is a temperature-enhanced opportunistic pathogen of E. huxleyi. We also detected caspase-like activity in dying C type cells co-cultured with R11, which suggests that programmed cell death plays a role in the death of E. huxleyi triggered by R11 - a mechanism induced by viruses (EhVs) and implicated in E. huxleyi bloom collapse. Given that E. huxleyi has recently been

  10. Detection of Biomolecular Binding Through Enhancement of Localized Surface Plasmon Resonance (LSPR by Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Min-Gon Kim

    2009-03-01

    Full Text Available To amplify the difference in localized surface plasmon resonance (LSPR spectra of gold nano-islands due to intermolecular binding events, gold nanoparticles were used. LSPR-based optical biosensors consisting of gold nano-islands were readily made on glass substrates using evaporation and heat treatment. Streptavidin (STA and biotinylated bovine serum albumin (Bio-BSA were chosen as the model receptor and the model analyte, respectively, to demonstrate the effectiveness of this detection method. Using this model system, we were able to enhance the sensitivity in monitoring the binding of Bio-BSA to gold nano-island surfaces functionalized with STA through the addition of gold nanoparticle-STA conjugates. In addition, SU-8 well chips with gold nano-island surfaces were fabricated through a conventional UV patterning method and were then utilized for image detection using the attenuated total reflection mode. These results suggest that the gold nano-island well chip may have the potential to be used for multiple and simultaneous detection of various bio-substances.

  11. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2015-11-19

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences.

  12. Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

    Directory of Open Access Journals (Sweden)

    Caitlin Siobhan Byrt

    2012-11-01

    Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

  13. Measuring binding kinetics of aromatic thiolated molecules with nanoparticles via surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Devetter, Brent M.; Mukherjee, Prabuddha; Murphy, Catherine J.; Bhargava, Rohit

    2015-05-01

    Colloidal plasmonic nanomaterials, consisting of metals such as gold and silver, are excellent candidates for advanced optical probes and devices, but precise control over surface chemistry is essential for realizing their full potential. Coupling thiolated (R-SH) molecules to nanoprobe surfaces is a convenient and established route to tailor surface properties. The ability to dynamically probe and monitor the surface chemistry of nanoparticles in solution is essential for rapidly manufacturing spectroscopically tunable nanoparticles. In this study, we report the development of surface-enhanced Raman spectroscopy (SERS) as a method to monitor the kinetics of gold-thiolate bond formation on colloidal gold nanoparticles. A theoretical model combining SERS enhancement with the Beer-Lambert law is proposed to explain ensemble scattering and absorption effects in colloids during chemisorption. In order to maximize biological relevance and signal reproducibility, experiments used to validate the model focused on maintaining nanoparticle stability after the addition of water-soluble aromatic thiolated molecules. Our results indicate that ligand exchange on gold nanoparticles follow a first-order Langmuir adsorption model with rate constants on the order of 0.01 min-1. This study demonstrates an experimental spectroscopic method and theoretical model for monitoring binding kinetics that may prove useful for designing novel probes.Colloidal plasmonic nanomaterials, consisting of metals such as gold and silver, are excellent candidates for advanced optical probes and devices, but precise control over surface chemistry is essential for realizing their full potential. Coupling thiolated (R-SH) molecules to nanoprobe surfaces is a convenient and established route to tailor surface properties. The ability to dynamically probe and monitor the surface chemistry of nanoparticles in solution is essential for rapidly manufacturing spectroscopically tunable nanoparticles. In this

  14. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

    Directory of Open Access Journals (Sweden)

    Jonathan Göke

    2011-12-01

    Full Text Available Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

  15. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA

    OpenAIRE

    Smith, Janet L.; Grossman, Alan D.

    2015-01-01

    DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep ...

  16. Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse

    OpenAIRE

    Kimura Shioko; Paiz Jorge; Yoneda Mitsuhiro; Kido Taketomo; Vinson Charles; Ward Jerrold M

    2012-01-01

    Abstract Background The CCAAT/enhancer binding proteins (C/EBPs) play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known. Methods A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP) gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino)-1-(3-pyri...

  17. Quorum-Sensing Blockade As A Strategy for Enhancing Host Defences Against Bacterial Pathogens

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Givskov, Michael Christian

    2007-01-01

    Conventional antibiotics target the growth and the basal life processes of bacteria leading to growth arrest and cell death. The selective force that is inherently linked to this mode of action eventually selects out antibiotic-resistant variants. The most obvious alternative to antibiotic...... is likely to increase the susceptibility of the infecting organism to host defences and its clearance from the host. The use of QS signal blockers to attenuate bacterial pathogenicity, rather than bacterial growth, is therefore highly attractive, particularly with respect to the emergence of multi-antibiotic...... resistant bacteria....

  18. Identification of an unconventional E3 binding surface on the UbcH5 Ub conjugate recognized by a pathogenic bacterial E3 ligase.

    Energy Technology Data Exchange (ETDEWEB)

    Levin, I.; Eakin, C.; Blanc, M. -P.; Klevit, R. E.; Miller, S. I.; Brzovic, P. S.

    2010-02-01

    Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5Ubn). Rapid generation of UbcH5Ubn may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

  19. Identification of an unconventional E3 binding surface on the UbcH5 ~ Ub conjugate recognized by a pathogenic bacterial E3 ligase.

    Science.gov (United States)

    Levin, Itay; Eakin, Catherine; Blanc, Marie-Pierre; Klevit, Rachel E; Miller, Samuel I; Brzovic, Peter S

    2010-02-16

    Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ~ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ~ Ub(n)). Rapid generation of UbcH5 ~ Ub(n) may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction. PMID:20133640

  20. Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

    Directory of Open Access Journals (Sweden)

    Kiyotaka Sakai

    2009-10-01

    Full Text Available Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A caused by the binding with immunoglobulin G (IgG in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

  1. Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media▿

    Science.gov (United States)

    Furukawa, Masayuki; Ebmeyer, Jörg; Pak, Kwang; Austin, Darrell A.; Melhus, Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2007-01-01

    Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation. PMID:17325051

  2. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages

    OpenAIRE

    1989-01-01

    Lipopolysaccharide binding protein (LBP) is an acute-phase reactant that binds bacterial LPS. We show that LBP binds to the surface of live Salmonella and to LPS coated erythrocytes (ELPS), and strongly enhances the attachment of these particles to macrophages. LBP bridges LPS- coated particles to macrophages (MO) by first binding to the LPS, then binding to MO. Pretreatment of ELPS with LBP enabled binding to MO, but pretreatment of MO had no effect. Moreover, MO did not recognize erythrocyt...

  3. Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens

    International Nuclear Information System (INIS)

    Nano-TiO2 is immunotoxic to fish and reduces the bactericidal function of fish neutrophils. Here, fathead minnows (Pimephales promelas) were exposed to low and high environmentally relevant concentration of nano-TiO2 (2 ng g−1 and 10 μg g−1 body weight, respectively), and were challenged with common fish bacterial pathogens, Aeromonas hydrophila or Edwardsiella ictaluri. Pre-exposure to nano-TiO2 significantly increased fish mortality during bacterial challenge. Nano-TiO2 concentrated in the kidney and spleen. Phagocytosis assay demonstrated that nano-TiO2 has the ability to diminish neutrophil phagocytosis of A. hydrophila. Fish injected with TiO2 nanoparticles displayed significant histopathology when compared to control fish. The interplay between nanoparticle exposure, immune system, histopathology, and infectious disease pathogenesis in any animal model has not been described before. By modulating fish immune responses and interfering with resistance to bacterial pathogens, manufactured nano-TiO2 has the potential to affect fish survival in a disease outbreak. - Highlights: • First data on the effect of nano-TiO2 pre-exposure on responses to bacterial pathogens. • Interplay between nano-TiO2, immune system, histopathology, and bacteria is described. • Nano-TiO2 has the potential to affect fish population survival in a disease outbreak. - By modulating fish immune responses and interfering with resistance to bacterial pathogens, internalized environmentally relevant concentrations of nano-TiO2 have potential to increase mortality of fish exposed to infectious disease challenge

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

    International Nuclear Information System (INIS)

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

  5. Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

    Directory of Open Access Journals (Sweden)

    Laura A. Novotny

    2016-08-01

    Full Text Available The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.

  6. Structure-Based Rational Design to Enhance the Solubility and Thermostability of a Bacterial Laccase Lac15

    Science.gov (United States)

    Fang, Zemin; Zhou, Peng; Chang, Fei; Yin, Qiang; Fang, Wei; Yuan, Jing; Zhang, Xuecheng; Xiao, Yazhong

    2014-01-01

    Bacterial laccases are ideal alternatives of fungal laccases for specific industrial applications due to specific characteristics such as alkalescence dependence and high chloride tolerance. However, some bacterial laccases presented as inclusion bodies when expressing in Escherichia coli and showed thermal instability. In this study, rational design was employed to enhance the solubility and the thermostablity of the bacterial laccase Lac15-His6 based on the crystal structure obtained previously. After deletion of His-tag and residues323–332, the obtained Lac15D was completely expressed in soluble form even at a higher temperature of 28°C, compared to only 50% of Lac15-His6 expressed solubly at 16°C. It showed a two-time higher activity at temperatures lower than 35°C and a half-life increasing from 72 min to 150 min at 45°C. When used in chromogenic reactions, Lac15D showed constant activity toward dye precursors and their combinations under alkaline conditions, demonstrating its application potential in hair coloring biotechnology. PMID:25036001

  7. Structure-based rational design to enhance the solubility and thermostability of a bacterial laccase Lac15.

    Directory of Open Access Journals (Sweden)

    Zemin Fang

    Full Text Available Bacterial laccases are ideal alternatives of fungal laccases for specific industrial applications due to specific characteristics such as alkalescence dependence and high chloride tolerance. However, some bacterial laccases presented as inclusion bodies when expressing in Escherichia coli and showed thermal instability. In this study, rational design was employed to enhance the solubility and the thermostablity of the bacterial laccase Lac15-His6 based on the crystal structure obtained previously. After deletion of His-tag and residues323-332, the obtained Lac15D was completely expressed in soluble form even at a higher temperature of 28°C, compared to only 50% of Lac15-His6 expressed solubly at 16°C. It showed a two-time higher activity at temperatures lower than 35°C and a half-life increasing from 72 min to 150 min at 45°C. When used in chromogenic reactions, Lac15D showed constant activity toward dye precursors and their combinations under alkaline conditions, demonstrating its application potential in hair coloring biotechnology.

  8. Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Givskov, Michael Christian

    2007-01-01

    Conventional antibiotics target the growth and the basal life processes of bacteria leading to growth arrest and cell death. The selective force that is inherently linked to this mode of action eventually selects out antibiotic-resistant variants. The most obvious alternative to antibiotic...... rise to a new 'drug target rush'. Recently, QS has been shown to be involved in the development of tolerance to various antimicrobial treatments and immune modulation. The regulation of virulence via QS confers a strategic advantage over host defences. Consequently, a drug capable of blocking QS...... is likely to increase the susceptibility of the infecting organism to host defences and its clearance from the host. The use of QS signal blockers to attenuate bacterial pathogenicity, rather than bacterial growth, is therefore highly attractive, particularly with respect to the emergence of multi-antibiotic...

  9. Selection and breeding of corn to enhance associative bacterial nitrogen fixation

    Energy Technology Data Exchange (ETDEWEB)

    Ela, S.W.; Anderson, M.A.; Brill, W.J.

    1980-01-01

    We have increased, through screening and breeding, the ability of corn (maize, Zea mays L.) to support bacterial nitrogen fixation in or on corn roots. Isotopic N fixed from /sup 15/N/sub 2/ was found on the roots. Even though the nitrogen-fixing association depends on germ plasm from tropical corn, the activity can be bred into corn currently used in midwestern United States agriculture.

  10. Selection and breeding of corn to enhance associative bacterial nitrogen fixation

    International Nuclear Information System (INIS)

    We have increased, through screening and breeding, the ability of corn (maize, Zea mays L.) to support bacterial nitrogen fixation in or on corn roots. Isotopic N fixed from 15N2 was found on the roots. Even though the nitrogen-fixing association depends on germ plasm from tropical corn, the activity can be bred into corn currently used in midwestern United States agriculture

  11. Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation

    OpenAIRE

    Röling, Wilfred F. M.; Milner, Michael G.; Jones, D. Martin; Lee, Kenneth; Daniel, Fabien; Swannell, Richard J. P.; Head, Ian M.

    2002-01-01

    Degradation of oil on beaches is, in general, limited by the supply of inorganic nutrients. In order to obtain a more systematic understanding of the effects of nutrient addition on oil spill bioremediation, beach sediment microcosms contaminated with oil were treated with different levels of inorganic nutrients. Oil biodegradation was assessed respirometrically and on the basis of changes in oil composition. Bacterial communities were compared by numerical analysis of denaturing gradient gel...

  12. Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice

    Science.gov (United States)

    Verma, Rajkumar; Venna, Venugopal R.; Liu, Fudong; Chauhan, Anjali; Koellhoffer, Edward; Patel, Anita; Ricker, Austin; Maas, Kendra; Graf, Joerg; McCullough, Louise D.

    2016-01-01

    Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke. PMID:27115295

  13. Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance of Microbial Electrochemical Systems Using Nano-Decorated Anodes

    Science.gov (United States)

    Xu, Shoutao

    Microbial electrochemical systems (MESs) have attracted much research attention in recent years due to their promising applications in renewable energy generation, bioremediation, and wastewater treatment. In a MES, microorganisms interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the cathode. The bacterial community of a high power mixed consortium MESs (maximum power density is 6.5W/m2) was analyzed by using denature gradient gel electrophoresis (DGGE) and 16S DNA clone library methods. The bacterial DGGE profiles were relatively complex (more than 10 bands) but only three brightly dominant bands in DGGE results. These results indicated there are three dominant bacterial species in mixed consortium MFCs. The 16S DNA clone library method results revealed that the predominant bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter sp. These three bacterial species reached to 88% of total bacterial species. This result is consistent with the DGGE result which showed that three bright bands represented three dominant bacterial species. Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel cell by conventional plating techniques with ferric citrate as electron acceptor under anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the most closely related species. The bacterial strain SX-1 produced electricity from citrate, acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density of 205 mA/m2 generated from citrate. Cyclic voltammetry analysis indicated that membrane associated proteins may play an important role in facilitating electron transfer from the bacteria to the electrode. This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors

  14. Bacterial cellulose-polyaniline nano-biocomposite: A porous media hydrogel bioanode enhancing the performance of microbial fuel cell

    Science.gov (United States)

    Mashkour, Mehrdad; Rahimnejad, Mostafa; Mashkour, Mahdi

    2016-09-01

    Microbial fuel cells (MFCs) are one of the possible renewable energy supplies which microorganisms play an active role in bio-oxidize reactions of a substrate such as glucose. Electrode materials and surface modifications are highly effective tools in enhancing MFCs' Performance. In this study, new composite anodes are fabricated. Bacterial cellulose (BC) is used as continuous phase and polyaniline (PANI) as dispersed one which is synthesized by in situ chemical oxidative polymerization on BC's fibers. With hydrogel nature of BC as a novel feature and polyaniline conductivity there meet the favorable conditions to obtain an active microbial biofilm on anode surface. Maximum power density of 117.76 mW/m2 in current density of 617 mA/m2 is achieved for BC/PANI anode. The amounts demonstrate a considerable enhancement compared with graphite plate (1 mW/m2 and 10 mA/m2).

  15. Zinc-oxide-silica-silver nanocomposite: Unique one-pot synthesis and enhanced catalytic and anti-bacterial performance.

    Science.gov (United States)

    Kokate, Mangesh; Garadkar, Kalyanrao; Gole, Anand

    2016-12-01

    We describe herein a unique approach to synthesize zinc oxide-silica-silver (ZnO-SiO2-Ag) nanocomposite, in a simple, one-pot process. The typical process for ZnO synthesis by alkaline precipitation of zinc salts has been tweaked to replace alkali by alkaline sodium silicate. The free acid from zinc salts helps in the synthesis of silica nanoparticles, whereas the alkalinity of sodium silicate precipitates the zinc salts. Addition of silver ions into the reaction pot prior to addition of sodium silicate, and subsequent reduction by borohydride, gives additional functionality of metallic centres for catalytic applications. The synthesis strategy is based on our recent work typically involving acid-base type of cross-reactions and demonstrates a novel strategy to synthesize nanocomposites in a one-pot approach. Each component in the composite offers a unique feature. ZnO besides displaying mild catalytic and anti-bacterial behaviour is an excellent and a cheap 3-D support for heterogeneous catalysis. Silver nanoparticles enhance the catalytic & anti-bacterial properties of ZnO. Silica is an important part of the composite; which not only "glues" the two nanoparticles thereby stabilizing the nanocomposite, but also significantly enhances the surface area of the composite; which is an attractive feature of any catalyst composite. The nanocomposite is found to show excellent catalytic performance with very high turnover frequencies (TOFs) when studied for catalytic reduction of Rhodamine B (RhB) and 4-Nitrophenol (4-NP). Additionally, the composite has been tested for its anti-bacterial properties on three different bacterial strains i.e. E. coli, B. Cereus and Bacillus firmus. The mechanism for enhancement of catalytic performance has been probed by understanding the role of silica in offering accessibility to the catalyst via its porous high surface area network. The nanocomposite has been characterized by a host of different analytical techniques. The uniqueness of

  16. Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

    Science.gov (United States)

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  17. Hsp90-binding immunophilin FKBP51 forms complexes with hTERT enhancing telomerase activity.

    Science.gov (United States)

    Lagadari, Mariana; Zgajnar, Nadia R; Gallo, Luciana I; Galigniana, Mario D

    2016-08-01

    FK506-binding proteins are members of the immunophilin family of proteins. Those immunophilins associated to the 90-kDa-heat-shock protein, Hsp90, have been proposed as potential modulators of signalling cascade factors chaperoned by Hsp90. FKBP51 and FKBP52 are the best characterized Hsp90-bound immunophilins first described associated to steroid-receptors. The reverse transcriptase subunit of telomerase, hTERT, is also an Hsp90 client-protein and is highly expressed in cancer cells, where it is required to compensate the loss of telomeric DNA after each successive cell division. Because FKBP51 is also a highly expressed protein in cancer tissues, we analyzed its potential association with hTERT·Hsp90 complexes and its possible biological role. In this study it is demonstrated that both immunophilins, FKBP51 and FKBP52, co-immunoprecipitate with hTERT. The Hsp90 inhibitor radicicol disrupts the heterocomplex and favors the partial cytoplasmic relocalization of hTERT in similar manner as the overexpression of the TPR-domain peptide of the immunophilin. While confocal microscopy images show that FKBP51 is primarily localized in mitochondria and hTERT is totally nuclear, upon the onset of oxidative stress, FKBP51 (but not FKBP52) becomes mostly nuclear colocalizing with hTERT, and longer exposure times to peroxide favors hTERT export to mitochondria. Importantly, telomerase activity of hTERT is significantly enhanced by FKBP51. These observations support the emerging role assigned to FKBP51 as antiapoptotic factor in cancer development and progression, and describe for the first time the potential role of this immunophilin favoring the clonal expansion by enhancing telomerase activity. PMID:27233944

  18. Pili Binding to Asialo-GM1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalization by Pseudomonas aeruginosa

    OpenAIRE

    Comolli, James C; Waite, Leslie L.; Keith E Mostov; Joanne N. Engel

    1999-01-01

    The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to...

  19. Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads.

    Science.gov (United States)

    Chiang, Chung-Jen; Wang, Jen-You; Chen, Po-Ting; Chao, Yun-Peng

    2009-03-01

    Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads. PMID:19018526

  20. Myocyte-specific enhancer binding factor 2A expression is downregulated during temporal lobe epilepsy.

    Science.gov (United States)

    Huang, Yunyi; Wu, Xuling; Guo, Jing; Yuan, Jinxian

    2016-09-01

    Myocyte-specific enhancer binding factor 2A (MEF2A) is a multifunctional nuclear protein that regulates synaptogenesis, dendritic morphogenesis, and neuronal survival. This study aimed to investigate the expression pattern of MEF2A in epileptogenic processes. MEF2A expression was detected in 20 temporal neocortex tissue samples from patients with temporal lobe epilepsy (TLE) and 20 samples from trauma patients without epilepsy by real-time quantitative polymerase chain reaction, immunohistochemistry, double-label immunofluorescent staining, and western blot analysis. In addition, the expression patterns of MEF2A in the hippocampus and adjacent cortex of a lithium-pilocarpine-induced TLE rat model and control rats were examined. MEF2A was found to be expressed in the nuclei of neurons but not in the dendrites of neurons and astrocytes. MEF2A expression was significantly downregulated in temporal neocortex of humans and rats with TLE compared to the control groups. In addition, in the lithium-pilocarpine-induced TLE model, MEF2A expression dynamically decreased within 2 months. Taken together, these data suggest that MEF2A is involved in the pathogenesis of TLE. PMID:26439092

  1. Debranching of soluble wheat arabinoxylan dramatically enhances recalcitrant binding to cellulose

    DEFF Research Database (Denmark)

    Selig, Michael J.; Thygesen, Lisbeth G.; Felby, Claus;

    2015-01-01

    The presence of xylan is a detriment to the enzymatic saccharification of cellulose in lignocelluloses. The inhibition of the processive cellobiohydrolase Cel7A by soluble wheat arabinoxylan is shown here to increase by 50 % following enzymatic treatment with a commercially-purified α-l-arabinofu......The presence of xylan is a detriment to the enzymatic saccharification of cellulose in lignocelluloses. The inhibition of the processive cellobiohydrolase Cel7A by soluble wheat arabinoxylan is shown here to increase by 50 % following enzymatic treatment with a commercially-purified α......-l-arabinofuranosidase. The enhanced inhibitory effect was shown by T2 relaxation time measurements via low field NMR to coincide with an increasing degree of constraint put on the water in xylan solutions. Furthermore, quartz crystal micro-balance with dissipation experiments showed that α-l-arabinofuranosidase treatment...... considerably increased the rate and rigidity of arabinoxylan mass association with cellulose. These data also suggest significant xylan–xylan adlayer formation occurs following initial binding of debranched arabinoxylan. From this, we speculate the inhibitory effects of xylan to cellulases may result from...

  2. Expression and Critical Role of Interleukin Enhancer Binding Factor 2 in Hepatocellular Carcinoma

    Science.gov (United States)

    Cheng, Shaobing; Jiang, Xu; Ding, Chaofeng; Du, Chengli; Owusu-Ansah, Kwabena Gyabaah; Weng, Xiaoyu; Hu, Wendi; Peng, Chuanhui; Lv, Zhen; Tong, Rongliang; Xiao, Heng; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2016-01-01

    Interleukin enhancer binding factor 2 (ILF2), a transcription factor, regulates cell growth by inhibiting the stabilization of mRNA. Currently, its role has gained recognition as a factor in the tumorigenic process. However, until now, little has been known about the detailed role ILF2 plays in hepatocellular carcinoma (HCC). In this study, we investigated the expression levels of ILF2 in HCC tissue with Western blot and immunohistochemical assays. To examine the effect of ILF2 on liver cancer cell growth and apoptosis, small interfering RNAs (siRNAs) targeting ILF2 were recombined to create lentiviral overexpression vectors. Our results showed higher expression levels of ILF2 mRNA and ILF2 protein in HCC tissue compared with matched peritumoral tissue. Expression of ILF2 may regulate cell growth and apoptosis in liver cancer cells via regulation of B-cell lymphoma 2 (Bcl-2), Bcl-2 related ovarian killer (Bok), Bcl-2-associated X protein (BAX), and cellular inhibitor of apoptosis 1 (cIAP1). Moreover, we inoculated nude mice with liver cancer cells to investigate the effect of ILF2 on tumorigenesis in vivo. As expected, a rapid growth was observed in cancer cells inoculated with a lentiviral vector coding Flag-ILF2 (Lenti-ILF2) compared with the control cells. Hence, these results promote a better understanding of ILF2’s potential role as a therapeutic target in HCC. PMID:27556459

  3. Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

    Science.gov (United States)

    Money, T; Jones, T; Dixon, R; Austin, S

    1999-08-01

    In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. PMID:10419940

  4. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    Science.gov (United States)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  5. Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso (NWU); (SKI)

    2010-10-22

    Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

  6. Transcription factor CCAAT/enhancer binding protein alpha up-regulates microRNA let-7a-1 in lung cancer cells by direct binding

    OpenAIRE

    Lin, Yani; Zhao, Jian; Hu, Xiaoyan; Wang, Lina; Liang, Liming; Chen, Weiwen

    2016-01-01

    Aims The transcription factor CCAAT/enhancer binding protein α (C/EBPα) and microRNA (miRNA) let-7a-1 act as tumor suppressors in many types of cancers including lung cancer. In the present study, we aim to investigate whether let-7a-1 is a novel important target of C/EBPα in lung cancer cells. Methods The DNA sequence of the 2.1 kb let-7a-1 promoter was analyzed with MatInspector 4.1 (http://www.genomatix.de). Human lung cancer cell lines A549 and H1299, and human cervical cancer cell line H...

  7. Enhancement of [123I[β-CIT binding in the striatum with clomipramine: is there a serotonin-dopamine interaction?

    International Nuclear Information System (INIS)

    Many reports support the concept of serotonergic-dopaminergic interaction in the brain. However, at present, there are few methods to study this relationship in vivo. The purpose of this study was to investigate the effect of serotonin (5-HT) uptake inhibitor, clomipramine, on a dopamine (DA) transporter ligand, [123I[β-CIT (RTI-55), in rat brain. Dose-dependent changes in [123I[β-CIT specific binding induced by clomipramine were studied in the striatum (rich in DA transporter) and the hypothalamus (rich in 5-HT transporter). The changes in the time-activity curves of [123I[β-CIT specific binding after clomipramine injection were also examined in these two regions. Using the cerebellum as the reference region, k3and k 4values with and without clomipramine administration were estimated by a two-compartment kinetic analysis. Clomipramine inhibited [123I[β-CIT specific binding in the hypothalamus, but enhanced its specific binding in the striatum in a dose-dependent manner. Kinetic analysis showed that k 3in the striatum was increased by 55%. In conclusion, enhancement of [123I[fbeta-CIT binding in the striatum after clomipramine administration indicated the possibility of 5-HT-DA interaction. (orig.). With 3 figs., 1 tab

  8. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    Science.gov (United States)

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-01

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

  9. Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens.

    Science.gov (United States)

    Li, Lin-Xi; Benoun, Joseph M; Weiskopf, Kipp; Garcia, K Christopher; McSorley, Stephen J

    2016-10-01

    Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

  10. Use of bacterial extracts to enhance amino acid breakdown in dry fermented sausages.

    Science.gov (United States)

    Herranz, B; Fernández, M; de la Hoz, L; Ordóñez, J A

    2006-02-01

    The effect of the intracellular cell-free extracts (ICFEs) of two bacterial strains (Lactobacillus sakei GO and Bacillus pumilus) on the amino acid catabolism and the sensory properties of dry fermented sausages, was investigated. Extracts were added to sausages alone or in combination with a protease, papain. Amino acid breakdown was monitored by the changes in free amino acids, ammonia and amine content during the ripening process. A 15% decrease in the content of free amino acids was observed in sausages added with the ICFE from L. sakei GO. Furthermore, the extract of L. sakei GO significantly reduced (54-68%) the content of the amino acids considered as precursors of the typical ripened flavour, i.e., valine, leucine and isoleucine. Chemical changes were not reflected in a significant improvement of the sensory quality of sausages added with the ICFEs. The potential use of the bacterial ICFEs studied in the present work for the manufacture of dry fermented sausages, and its comparison with the use of fungal extracts, are discussed. PMID:22061560

  11. Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.

    Science.gov (United States)

    Johnson, M Brittany; Criss, Alison K

    2013-08-01

    Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  12. Spermidine/spermine N-1-acetyltransferase specifically binds to the integrin alpha 9 subunit cytoplasmic domain and enhances cell migration

    OpenAIRE

    Chen, C.; Young, B A; Coleman, C S; Pegg, A E; Sheppard, D

    2004-01-01

    T he integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the beta cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-h...

  13. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    Science.gov (United States)

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

  14. Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse

    Directory of Open Access Journals (Sweden)

    Kimura Shioko

    2012-12-01

    Full Text Available Abstract Background The CCAAT/enhancer binding proteins (C/EBPs play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known. Methods A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined. Results A-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse. Conclusions The DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.

  15. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  16. High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

    Science.gov (United States)

    Swain, Monalisa; Slomiany, Mark G.; Rosenzweig, Steven A.; Atreya, Hanudatta S.

    2010-01-01

    The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-IR). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1–6; 22–31 kDa) that via high affinity binding to the IGFs (KD ~ 300–700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in E. coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP. PMID:20541521

  17. Automated discovery of tissue-targeting enhancers and transcription factors from binding motif and gene function data.

    Directory of Open Access Journals (Sweden)

    Geetu Tuteja

    2014-01-01

    Full Text Available Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86% tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87% tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.

  18. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

    Institute of Scientific and Technical Information of China (English)

    Paul C. SEHNKE; Beth J. LAUGHNER; Carla R. LYERLY LINEBARGER; William B. GURLEY; Robert J.FERL

    2005-01-01

    Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

  19. Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome

    Science.gov (United States)

    Luo, Xi; Wasilko, David J.; Liu, Yao; Sun, Jiayi; Wu, Xiaochun; Luo, Zhao-Qing; Mao, Yuxin

    2015-01-01

    The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires’ disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells. PMID

  20. Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome.

    Directory of Open Access Journals (Sweden)

    Xi Luo

    2015-06-01

    Full Text Available The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV. SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871. The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4P binding of SidC comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4P from a closed form to an open active form. Mutations of key residues involved in PI(4P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4P probe in living cells.

  1. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    International Nuclear Information System (INIS)

    Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni2+ ions but that it is able to bind Zn2+ with Kd < 70 nM. It is concluded that Zn2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors

  2. Crystal structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Meiying; Cooper, David; Grossoehmerb, Nickolas; Yu, Minmin; Hung, Li-Wei; Cieslik, Murcin; Derewendaro, Urszula; Lesley, Scott; Wilson, Ian; Giedrocb, David; Derewenda, Zygmunt

    2009-06-06

    The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged helix (WH) DNA-binding domains and diverse C-terminal, regulatory domains, which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all a-helical regulatory domains classified into two related Pfam families: FadR{_}C and FCD. Only two crystal structures of the FadR family members, i.e. the E. coli FadR protein and the LldR from C. glutamicum, have been described to date in literature. Here we describe the crystal structure of TM0439, a GntR regulator with an FCD domain, found in the Thermotoga maritima genome. The FCD domain is similar to that of the LldR regulator, and contains a buried metal binding site. Using atomic absorption spectroscopy and Trp fluorescence, we show that the recombinant protein contains bound Ni{sup 2+} ions, but it is able to bind Zn{sup 2+} with K{sub D} < 70 nM . We conclude that Zn{sup 2+} is the likely physiological metal, where it may perform either or both structural and regulatory roles. Finally, we compare the TM0439 structure to two other FadR family structures recently deposited by Structural Genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

  3. Yeast One-hybrid System Used to Identify the Binding Proteins for Rat Glutathione S-transferase P Enhancer I

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Methods Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. Results cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated, The binding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

  4. CCAAT/enhancer binding protein β-deficiency enhances type 1 diabetic bone phenotype by increasing marrow adiposity and bone resorption

    OpenAIRE

    Motyl, Katherine J.; Raetz, Michelle; Tekalur, Srinivasan Arjun; Schwartz, Richard C.; McCabe, Laura R.

    2011-01-01

    Bone loss in type 1 diabetes is accompanied by increased marrow fat, which could directly reduce osteoblast activity or result from altered bone marrow mesenchymal cell lineage selection (adipocyte vs. osteoblast). CCAAT/enhancer binding protein beta (C/EBPβ) is an important regulator of both adipocyte and osteoblast differentiation. C/EBPβ-null mice have delayed bone formation and defective lipid accumulation in brown adipose tissue. To examine the balance of C/EBPβ functions in the diabetic...

  5. Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

    DEFF Research Database (Denmark)

    Nijnik, Anastasia; Madera, Laurence; Ma, Shuhua;

    2010-01-01

    With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial...... activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study......, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus...

  6. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  7. Enhancement of Methacholine-Evoked Tracheal Contraction Induced by Bacterial Lipopolysaccharides Depends on Epithelium and Tumor Necrosis Factor

    Directory of Open Access Journals (Sweden)

    T. Secher

    2012-01-01

    Full Text Available Inhaled bacterial lipopolysaccharides (LPSs induce an acute tumour necrosis factor-alpha (TNF-α- dependent inflammatory response in the murine airways mediated by Toll-like receptor 4 (TLR4 via the myeloid differentiation MyD88 adaptor protein pathway. However, the contractile response of the bronchial smooth muscle and the role of endogenous TNFα in this process have been elusive. We determined the in vivo respiratory pattern of C57BL/6 mice after intranasal LPS administration with or without the presence of increasing doses of methacholine (MCh. We found that LPS administration altered the basal and MCh-evoked respiratory pattern that peaked at 90 min and decreased thereafter in the next 48 h, reaching basal levels 7 days later. We investigated in controlled ex vivo condition the isometric contraction of isolated tracheal rings in response to MCh cholinergic stimulation. We observed that preincubation of the tracheal rings with LPS for 90 min enhanced the subsequent MCh-induced contractile response (hyperreactivity, which was prevented by prior neutralization of TNFα with a specific antibody. Furthermore, hyperreactivity induced by LPS depended on an intact epithelium, whereas hyperreactivity induced by TNFα was well maintained in the absence of epithelium. Finally, the enhanced contractile response to MCh induced by LPS when compared with control mice was not observed in tracheal rings from TLR4- or TNF- or TNF-receptor-deficient mice. We conclude that bacterial endotoxin-mediated hyperreactivity of isolated tracheal rings to MCh depends upon TLR4 integrity that signals the activation of epithelium, which release endogenous TNFα.

  8. Hypertonic saline enhances host response to bacterial challenge by augmenting receptor-independent neutrophil intracellular superoxide formation.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    OBJECTIVE: This study sought to determine whether hypertonic saline (HTS) infusion modulates the host response to bacterial challenge. METHODS: Sepsis was induced in 30 Balb-C mice by intraperitoneal injection of Escherichia coli (5 x 107 organisms per animal). In 10 mice, resuscitation was performed at 0 and 24 hours with a 4 mL\\/kg bolus of HTS (7.5% NaCl), 10 animals received 4 mL\\/kg of normal saline (0.9% NaCl), and the remaining animals received 30 mL\\/kg of normal saline. Samples of blood, spleen, and lung were cultured at 8 and 36 hours. Polymorphonucleocytes were incubated in isotonic or hypertonic medium before culture with E. coli. Phagocytosis was assessed by flow cytometry, whereas intracellular bacterial killing was measured after inhibition of phagocytosis with cytochalasin B. Intracellular formation of free radicals was assessed by the molecular probe CM-H(2)DCFDA. Mitogen-activated protein (MAP) kinase p38 and ERK-1 phosphorylation, and nuclear factor kappa B (NFkappaB) activation were determined. Data are represented as means (SEM), and an analysis of variance test was performed to gauge statistical significance. RESULTS: Significantly reduced bacterial culture was observed in the animals resuscitated with HTS when compared with their NS counterparts, in blood (51.8 +\\/- 4.3 vs. 82.0 +\\/- 3.3 and 78.4 +\\/- 4.8, P = 0.005), lung (40.0 +\\/- 4.1 vs. 93.2 +\\/- 2.1 and 80.9 +\\/- 4.7, P = 0.002), and spleen (56.4 +\\/- 3.8 vs. 85.4 +\\/- 4.2 and 90.1 +\\/- 5.9, P = 0.05). Intracellular killing of bacteria increased markedly (P = 0.026) and superoxide generation was enhanced upon exposure to HTS (775.78 +\\/- 23.6 vs. 696.57 +\\/- 42.2, P = 0.017) despite inhibition of MAP kinase and NFkappaB activation. CONCLUSIONS: HTS significantly enhances intracellular killing of bacteria while attenuating receptor-mediated activation of proinflammatory cascades.

  9. Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

    OpenAIRE

    Aly, Rabab M.; Ansaf B. Yousef

    2015-01-01

    Objective: Lymphoid enhancer-binding factor-1 (LEF-1) is a key transcription factor of wingless-type (Wnt) signaling in various tumors and it is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF-1 in acute lymphoblastic leukemia (ALL) and determined its specific prognostic significance in this disease. Materials and Methods: We studied LEF-1 expression in 56 newly diagnosed B-acute ALL adult patients using real-time quantitative polymer...

  10. Optimized Gamma Synchronization Enhances Functional Binding of Fronto-Parietal Cortices in Mathematically Gifted Adolescents during Deductive Reasoning

    OpenAIRE

    ZHANG Li; Gan, John Q.; Wang, Haixian

    2014-01-01

    As enhanced fronto-parietal network has been suggested to support reasoning ability of math-gifted adolescents, the main goal of this EEG source analysis is to investigate the temporal binding of the gamma-band (30–60 Hz) synchronization between frontal and parietal cortices in adolescents with exceptional mathematical ability, including the functional connectivity of gamma neurocognitive network, the temporal dynamics of fronto-parietal network (phase-locking durations and network lability i...

  11. The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development.

    OpenAIRE

    Samuelsson, L; Strömberg, K; Vikman, K; Bjursell, G; Enerbäck, S

    1991-01-01

    During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP)...

  12. Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2006-01-01

    Aim:To investigate the effect of incorporation of all isoleucine zipper(IZ)motif into CD40 on binding activity of CD40 for the CD40 ligand (CD40L).Methods:Prokaryotic expression vectors for 2 soluble CD40 derivatives,shCD40His and shCD40IZ containing an IZ dowain,were constructed and expressed in Escherichia coli.The recombinant proteins were purified to homogeneity after refolding from inclusion bodies.Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography,and their binding activity for CD40L on Jurkat T cells was determined by flow cytometry.Results:shCD40His and shCD40IZ were generated.Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface.shCD40IZ had much higher binding activity to its ligand(CD40L)than did shCD40His.Furthermore,size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution.Conclusion:Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.

  13. Combining prebiotics with probiotic bacteria can enhance bacterial growth and secretion of bacteriocins.

    Science.gov (United States)

    Pranckutė, Raminta; Kaunietis, Arnoldas; Kuisienė, Nomeda; Čitavičius, Donaldas J

    2016-08-01

    There is a growing interest in supporting human health by using prebiotics, such as oligosaccharides, and beneficial bacteria, also called probiotics. Combining these two components we can develop synbiotics. In order to create successful combination of synbiotic it is very important to evaluate the influence of prebiotic oligosaccharides to probiotic bacteria and their behavior, such as growth and secretion of health related biomolecules, including bacteriocins. In this study seven type strains of probiotic bacteria (five Lactobacillus sp. and two Lactococcus sp.) and two Lactobacillus sp. strains, isolated from probiotic yoghurt, were cultivated with various commercially available and extracted oligosaccharides (OS). The aim of this study was to evaluate the influence of these OS on type and isolated bacterial strains growth and antibacterial activity. Obtained results suggest that combination of certain OS with probiotic strains may considerably improve their growth and/or antibacterial activity. We also determined the antibacterial activity spectrum of investigated strains with combination of OS against common food borne pathogens. Results of this work show that prebiotic OS can be useful for modulating probiotic bacteria growth, antibacterial activity and even specificity of this activity. PMID:27181578

  14. CHEMICALLY FABRICATED SILVER NANOPARTICLES ENHANCES THE ACTIVITY OF ANTIBIOTICS AGAINST SELECTED HUMAN BACTERIAL PATHOGENS

    Directory of Open Access Journals (Sweden)

    S. Thangapandiyan and P. Prema*

    2012-05-01

    Full Text Available Due to the outbreak of infectious diseases caused by different pathogenic bacteria and the development of antibiotic resistance, the pharmaceutical companies and the researchers are now searching for new unconventional antibacterial agents. Nanotechnology represents a modern and innovative approach to develop new formulations based on metallic nanoparticles with antimicrobial properties. The potential bioactivity of chemically fabricated silver nanoparticles has been extensively studied. However, the antibacterial activity of silver nanoparticles individually or in combination with different antibiotics has not been demonstrated. In the present investigations, the effect of silver nanoparticles on the antibacterial activity of different antibiotics was evaluated against selected human bacterial pathogens such as Staphylococcus aureus, Streptococcus epidermis, Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus by disc diffusion method. In the presence of sub - inhibitory concentration of silver nanoparticles (100µL/disc, the antibacterial activities of all antibiotics are increased from 1 mm to 10 mm. The maximum fold increase was noticed for vancomycin against Pseudomonas aeruginosa (66.67%, Escherichia coli (62.50%, and Staphylococcus aureus (46% followed by rifampicin against Bacillus cereus (66.67% and kanamycin against Streptococcus epidermis (25%. These results signify that the silver nanoparticles showed potential antibacterial action of ß-lactams, glycopeptides, aminoglycosides, sulphonamides suggesting a possible utilization of silver nanocompounds in combination therapy against selected pathogens used in the experiment.

  15. Bacterial fatty acids enhance recovery from the dauer larva in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Tiffany K Kaul

    Full Text Available The dauer larva is a specialized dispersal stage in the nematode Caenorhabditis elegans that allows the animal to survive starvation for an extended period of time. The dauer does not feed, but uses chemosensation to identify new food sources and to determine whether to resume reproductive growth. Bacteria produce food signals that promote recovery of the dauer larva, but the chemical identities of these signals remain poorly defined. We find that bacterial fatty acids in the environment augment recovery from the dauer stage under permissive conditions. The effect of increased fatty acids on different dauer constitutive mutants indicates a role for insulin peptide secretion in coordinating recovery from the dauer stage in response to fatty acids. These data suggest that worms can sense the presence of fatty acids in the environment and that elevated levels can promote recovery from dauer arrest. This may be important in the natural environment where the dauer larva needs to determine whether the environment is appropriate to support reproductive growth following dauer exit.

  16. Bacterial fatty acids enhance recovery from the dauer larva in Caenorhabditis elegans.

    Science.gov (United States)

    Kaul, Tiffany K; Reis Rodrigues, Pedro; Ogungbe, Ifedayo V; Kapahi, Pankaj; Gill, Matthew S

    2014-01-01

    The dauer larva is a specialized dispersal stage in the nematode Caenorhabditis elegans that allows the animal to survive starvation for an extended period of time. The dauer does not feed, but uses chemosensation to identify new food sources and to determine whether to resume reproductive growth. Bacteria produce food signals that promote recovery of the dauer larva, but the chemical identities of these signals remain poorly defined. We find that bacterial fatty acids in the environment augment recovery from the dauer stage under permissive conditions. The effect of increased fatty acids on different dauer constitutive mutants indicates a role for insulin peptide secretion in coordinating recovery from the dauer stage in response to fatty acids. These data suggest that worms can sense the presence of fatty acids in the environment and that elevated levels can promote recovery from dauer arrest. This may be important in the natural environment where the dauer larva needs to determine whether the environment is appropriate to support reproductive growth following dauer exit. PMID:24475206

  17. Superior hybrid hydrogels of polyacrylamide enhanced by bacterial cellulose nanofiber clusters.

    Science.gov (United States)

    Yuan, Ningxiao; Xu, Lu; Zhang, Lu; Ye, Haowen; Zhao, Jianhao; Liu, Zhong; Rong, Jianhua

    2016-10-01

    Hybrid polyacrylamide/bacterial cellulose nanofiber clusters (PAM/BC) hydrogels with high strength, toughness and recoverability were synthesized by in situ polymerization of acrylamide monomer in BC nanofiber clusters suspension. The hybrid gels exhibited an extremely large elongation at break of 2200%, and a high fracture stress of 1.35MPa. Additionally, the original length of hydrogels could be recovered after releasing the tensile force. Compressive results showed that the PAM/BC hybrid gels could reach a strain of about 99% without break, and was able to completely recover its original shape immediately after releasing the compression force. The compressive stress at 99% reached as high as 30MPa. Nearly no hysteresis in cyclic compressive tests was observed with these hybrid gels. The FT-IR, XRD and TGA analysis showed that hydrogen bonds between the PAM chains and BC nanofiber clusters mainly contributed to the superior mechanical properties of hybrid hydrogels. The cell viability results suggested that PAM/BC hybrid hydrogel was benign for biomedical application. These PAM/BC hydrogels offer a great promise as biomaterials such as bone and cartilage repair materials. PMID:27287117

  18. Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatibility complex class I genes

    NARCIS (Netherlands)

    Veer, L.J. van 't; Lutz, P.M.; Isselbacher, K.J.; Bernards, R.A.

    1992-01-01

    We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative

  19. The osmolyte xylitol reduces the salt concentration of airway surface liquid and may enhance bacterial killing

    Science.gov (United States)

    Zabner, Joseph; Seiler, Michael P.; Launspach, Janice L.; Karp, Philip H.; Kearney, William R.; Look, Dwight C.; Smith, Jeffrey J.; Welsh, Michael J.

    2000-10-01

    The thin layer of airway surface liquid (ASL) contains antimicrobial substances that kill the small numbers of bacteria that are constantly being deposited in the lungs. An increase in ASL salt concentration inhibits the activity of airway antimicrobial factors and may partially explain the pathogenesis of cystic fibrosis (CF). We tested the hypothesis that an osmolyte with a low transepithelial permeability may lower the ASL salt concentration, thereby enhancing innate immunity. We found that the five-carbon sugar xylitol has a low transepithelial permeability, is poorly metabolized by several bacteria, and can lower the ASL salt concentration in both CF and non-CF airway epithelia in vitro. Furthermore, in a double-blind, randomized, crossover study, xylitol sprayed for 4 days into each nostril of normal volunteers significantly decreased the number of nasal coagulase-negative Staphylococcus compared with saline control. Xylitol may be of value in decreasing ASL salt concentration and enhancing the innate antimicrobial defense at the airway surface.

  20. A Comparative Study of the Bacterial Community in Denitrifying and Traditional Enhanced Biological Phosphorus Removal Processes

    OpenAIRE

    Lv, Xiao-Mei; Shao, Ming-Fei; Li, Chao-Lin; Li, Ji; Gao, Xin-lei; Sun, Fei-Yun

    2014-01-01

    Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. Two lab-scale sequencing batch reactors (SBRs) were operated in alternating anaerobic-anoxic (A-A) or anaerobic-oxic (A-O) conditions to achieve denitrifying enhanced biological phosphate removal (EBPR) and traditional EBPR. No significant differences were observed in phosphorus removal efficiencies between A-A SBR and A-O SBR, with phosphoru...

  1. The osmolyte xylitol reduces the salt concentration of airway surface liquid and may enhance bacterial killing

    OpenAIRE

    Zabner, Joseph; Seiler, Michael P.; Launspach, Janice L.; Karp, Philip H.; Kearney, William R.; Look, Dwight C.; Smith, Jeffrey J.; Welsh, Michael J.

    2000-01-01

    The thin layer of airway surface liquid (ASL) contains antimicrobial substances that kill the small numbers of bacteria that are constantly being deposited in the lungs. An increase in ASL salt concentration inhibits the activity of airway antimicrobial factors and may partially explain the pathogenesis of cystic fibrosis (CF). We tested the hypothesis that an osmolyte with a low transepithelial permeability may lower the ASL salt concentration, thereby enhancing i...

  2. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    Science.gov (United States)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  3. Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    efficient with supercoiled than with linear DNA. In the presence of 140 mM KCI, the PNA binding rate was reduced but, notably, highly dependent on template topology. Negative supercoiling (mean superhelix density, sigma approximately -0.051) increased the rate of binding by 2 orders of magnitude compared...... to that of relaxed DNA. The pseudo-first-order rate constant [k psi (sigma)] obeys an exponential function, k psi (sigma) = k psi (lin)e-sigma delta, where delta is a constant of 105 and k psi lin is the rate of PNA binding to linear DNA (sigma = 0). The activation energy [Ea(sigma)] was determined as approximately...... 93 and approximately 48 kJ mol-1 for PNA binding to linear and supercoiled DNA, respectively. The results are discussed in relation to the possible future use of PNA as an antigene agent and in the framework of DNA "breathing" dynamics....

  4. Tubulin binding cofactor C (TBCC suppresses tumor growth and enhances chemosensitivity in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Laurier Jean-Fabien

    2010-04-01

    Full Text Available Abstract Background Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC, a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers. Methods We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells. Results TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to

  5. Contribution of the Collagen-Binding Proteins of Streptococcus mutans to Bacterial Colonization of Inflamed Dental Pulp

    Science.gov (United States)

    Nomura, Ryota; Ogaya, Yuko; Nakano, Kazuhiko

    2016-01-01

    Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (Pcanal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases. PMID:27442266

  6. Structure of the bacterial cell division determinant GpsB and its interaction with penicillin-binding proteins.

    Science.gov (United States)

    Rismondo, Jeanine; Cleverley, Robert M; Lane, Harriet V; Großhennig, Stephanie; Steglich, Anne; Möller, Lars; Mannala, Gopala Krishna; Hain, Torsten; Lewis, Richard J; Halbedel, Sven

    2016-03-01

    Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin-binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials. PMID:26575090

  7. Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections.

    Science.gov (United States)

    Baelo, Aida; Levato, Riccardo; Julián, Esther; Crespo, Anna; Astola, José; Gavaldà, Joan; Engel, Elisabeth; Mateos-Timoneda, Miguel Angel; Torrents, Eduard

    2015-07-10

    Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections. PMID:25913364

  8. Design of a minimal polypeptide unit for bacteriochlorophyll binding and self-assembly based on photosynthetic bacterial light-harvesting proteins.

    Science.gov (United States)

    Noy, Dror; Dutton, P Leslie

    2006-02-21

    We introduce LH1beta24, a minimal 24 amino acid polypeptide that binds and assembles bacteriochlorophylls (BChls) in micelles of octyl beta-glucoside (OG) into complexes with spectral properties that resemble those of B820, a universal intermediate in the assembly of native purple bacterial light-harvesting complexes (LHs). LH1beta24 was designed by a survey of sequences and crystal structures of bacterial LH proteins from different organisms combined with currently available information from in vitro reconstitution studies and genetically modified LHs in vivo. We took as a template for the design sphbeta31, a truncated 31 amino acid analogue of the native beta-apoprotein from the core LH complex of Rhodobacter sphaeroides. This peptide self-assembles with BChls to form B820 and, upon cooling and lowering OG concentration, forms red-shifted B850 spectral species that are considered analogous to native LH complexes. We find that LH1beta24 self-assembles with BChl in OG to form homodimeric B820-type subunits comprising two LH1beta24 and two BChl molecules per subunit. We demonstrate, by modeling the structure using the highly homologous structure of LH2 from Rhodospirillum molischianum, that it has the minimal size for BChl binding. Additionally, we have compared the self-assembly of sphbeta31 and LH1beta24 with BChls and discovered that the association enthalpies and entropies of both species are similar to those measured for native LH1 from Rhodospirillum rubrum. However, sphbeta31 readily aggregates into intermediate higher oligomeric species and further to form B850 species; moreover, the assembly process of these oligomers is not reversible, and they are apparently large nonspecific BChl-peptide coaggregates rather than well-defined nativelike LH complexes. Similar aggregates were observed during LH1beta24 assembly, but these were formed less readily and required lower temperatures than sphbeta31. In view of these results, we reevaluate previous in vitro

  9. Bacterial biosurfactants, and their role in microbial enhanced oil recovery (MEOR).

    Science.gov (United States)

    Khire, J M

    2010-01-01

    Surfactants are chemically synthesized surface-active compounds widely used for large number of applications in various industries. During last few years there is increase demand of biological surface-active compounds or biosurfactants which are produced by large number of microorganisms as they exert biodegradability, low toxicity and widespread application compared to chemical surfactants. They can be used as emulsifiers, de-emulsifiers, wetting agents, spreading agents, foaming agents, functional food ingredients and detergents. Various experiments at laboratory scale on sand-pack columns and field trials have successfully indicated effectiveness of biosurfactants in microbial enhanced oil recovery (MEOR). PMID:20545280

  10. Rapid identification of bacterial resistance to Ciprofloxacin using surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Kastanos, Evdokia; Hadjigeorgiou, Katerina; Pitris, Costas

    2014-02-01

    Due to its effectiveness and broad coverage, Ciprofloxacin is the fifth most prescribed antibiotic in the US. As current methods of infection diagnosis and antibiotic sensitivity testing (i.e. an antibiogram) are very time consuming, physicians prescribe ciprofloxacin before obtaining antibiogram results. In order to avoid increasing resistance to the antibiotic, a method was developed to provide both a rapid diagnosis and the sensitivity to the antibiotic. Using Surface Enhanced Raman Spectroscopy, an antibiogram was obtained after exposing the bacteria to Ciprofloxacin for just two hours. Spectral analysis revealed clear separation between sensitive and resistant bacteria and could also offer some inside into the mechanisms of resistance.

  11. Characterization of smart auto-degradative hydrogel matrix containing alginate lyase to enhance levofloxacin delivery against bacterial biofilms.

    Science.gov (United States)

    Islan, German A; Dini, Cecilia; Bartel, Laura C; Bolzán, Alejandro D; Castro, Guillermo R

    2015-12-30

    The aim of the present work is the characterization of smart auto-degradable microspheres composed of calcium alginate/high methoxylated pectin containing an alginate lyase (AL) from Sphingobacterium multivorum and levofloxacin. Microspheres were prepared by ionotropic gelation containing AL in its inactive form at pH 4.0. Incubation of microspheres in Tris-HCl and PBS buffers at pH 7.40 allowed to establish the effect of ion-chelating phosphate on matrix erodability and suggested an intrinsically activation of AL by turning the pH close to neutrality. Scanning electron and optical microscopies revealed the presence of holes and surface changes in AL containing microspheres. Furthermore, texturometric parameters, DSC profiles and swelling properties were showing strong changes in microspheres properties. Encapsulation of levofloxacin into microspheres containing AL showed 70% efficiency and 35% enhancement of antimicrobial activity against Pseudomonas aeruginosa biofilm. Levofloxacin release from microspheres was not changed at acidic pH, but was modified at neutral pH in presence of AL. Advantageously, only gel matrix debris were detectable after overnight incubation, indicating an autodegradative gel process activated by the pH. Absence of matrix cytotoxicity and a reduction of the levofloxacin toxicity after encapsulation were observed in mammalian CHO-K1 cell cultures. These properties make the system a potent and versatile tool for antibiotic oral delivery targeted to intestine, enhancing the drug bioavailability to eradicate bacterial biofilm and avoiding possible intestinal obstructions.

  12. Enhancement of microbial diversity and methane yield by bacterial bioaugmentation through the anaerobic digestion of Haematococcus pluvialis.

    Science.gov (United States)

    Aydin, Sevcan

    2016-06-01

    Microalgae has recalcitrant cell walls that may limit digestibility and, therefore, reduce bioenergy production. In light of the fact that cellulose can increase the cell wall recalcitrance of the Haematococcus pluvialis species of microalgae, the objective of this research was to examine how bioaugmentation with the Clostridium thermocellum at various inoculum ratios represents a viable method by which the CH4 production of microalgae can be enhanced. The results of the investigation revealed that bioaugmentation with C. thermocellum increased the degradation of H. pluvialis biomass and resulted in a 18-38 % increase in methane production as a result of increased cell disruption. In addition, the use of Illumina Miseq sequencing highlighted that the bacterial and archaeal diversity and quantities in the genus were enhanced as a result of the addition of C. thermocellum and this, in itself, improved the efficiency of the biodegradation. Bioaugmentation with C. thermocellum (%15) was also determined to represent the most energy-efficiency method of producing methane. PMID:27067588

  13. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    Science.gov (United States)

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

  14. Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells

    Directory of Open Access Journals (Sweden)

    Herpers Bjorn L

    2008-12-01

    Full Text Available Abstract Background Mannose binding lectin (MBL is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro. Results High avidity binding was observed between MBL and C. albicans and C. parapsilosis. Addition of MBL to MBL deficient serum increased the deposition of C4 and C3b and enhanced the uptake of C. albicans, C. parapsilosis and acapsular C. neoformans by polymorphonuclear cells (PMNs. Compared to other microorganisms, such as Escherichia coli, Staphylococcus aureus and Cryptococcus neoformans, C. parapsilosis and Candida albicans were potent activators of the lectin pathway. Conclusion Our results suggest that MBL plays a crucial role in the innate immunity against infections caused by yeast by increasing uptake by PMN.

  15. Listeria monocytogenes survival of UV-C radiation is enhanced by presence of sodium chloride, organic food material and by bacterial biofilm formation

    DEFF Research Database (Denmark)

    Bernbom, Nete; Vogel, Birte Fonnesbech; Gram, Lone

    2011-01-01

    a biofilm for 7days before exposure. It is not known if this enhanced survival is due to physiological changes in the attached bacterial cells, a physical protection of the cells in the food matrix or a combination. In conclusion, we demonstrate that UV-C light is a useful extra bacteriocidal step...

  16. Bacterial inoculants for enhanced seed germination of Spartina densiflora: Implications for restoration of metal polluted areas.

    Science.gov (United States)

    Paredes-Páliz, Karina I; Pajuelo, Eloísa; Doukkali, Bouchra; Caviedes, Miguel Ángel; Rodríguez-Llorente, Ignacio D; Mateos-Naranjo, Enrique

    2016-09-15

    The design of effective phytoremediation programs is severely hindered by poor seed germination on metal polluted soils. The possibility that inoculation with plant growth promoting rhizobacteria (PGPR) could help overcoming this problem is hypothesized. Our aim was investigating the role of PGPR in Spartina densiflora seed germination on sediments with different physicochemical characteristics and metal pollution degrees. Gram negative Pantoea agglomerans RSO6 and RSO7, and gram positive Bacillus aryabhattai RSO25, together with the consortium of the three strains, were used for independent inoculation experiments. The presence of metals (As, Cu, Pb and Zn) in sediments reduced seed germination by 80%. Inoculation with Bacillus aryabhattai RSO25 or Pantoea agglomerans RSO6 and RSO7 enhanced up to 2.5 fold the germination rate of S. densiflora in polluted sediments regarding non-inoculated controls. Moreover, the germination process was accelerated and the germination period was extended. The consortium did not achieve further improvements in seed germination. PMID:27315751

  17. A thermonuclease of Neisseria gonorrhoeae enhances bacterial escape from killing by neutrophil extracellular traps.

    Science.gov (United States)

    Juneau, Richard A; Stevens, Jacqueline S; Apicella, Michael A; Criss, Alison K

    2015-07-15

    Acute gonorrhea is characterized by neutrophilic inflammation that is insufficient to clear Neisseria gonorrhoeae. Activated neutrophils release extracellular traps (NETs), which are composed of chromatin and decorated with antimicrobial proteins. The N. gonorrhoeae NG0969 open reading frame contains a gene (nuc) that encodes a putatively secreted thermonuclease (Nuc) that contributes to biofilm remodeling. Here, we report that Nuc degrades NETs to help N. gonorrhoeae resist killing by neutrophils. Primary human neutrophils released NETs after exposure to N. gonorrhoeae, but NET integrity declined over time with Nuc-containing bacteria. Recombinant Nuc and conditioned medium from Nuc-containing N. gonorrhoeae degraded human neutrophil DNA and NETs. NETs were found to have antimicrobial activity against N. gonorrhoeae, and Nuc expression enhanced N. gonorrhoeae survival in the presence of neutrophils that released NETs. We propose that Nuc enables N. gonorrhoeae to escape trapping and killing by NETs during symptomatic infection, highlighting Nuc as a multifunctional virulence factor for N. gonorrhoeae.

  18. Enhanced inhibition of bacterial biofilm formation and reduced leukocyte toxicity by chloramphenicol:β-cyclodextrin:N-acetylcysteine complex.

    Science.gov (United States)

    Aiassa, Virginia; Zoppi, Ariana; Becerra, M Cecilia; Albesa, Inés; Longhi, Marcela R

    2016-11-01

    The purpose of this study was to improve the physicochemical and biological properties of chloramphenicol (CP) by multicomponent complexation with β-cyclodextrin (β-CD) and N-acetylcysteine (NAC). The present work describes the ability of solid multicomponent complex (MC) to decrease biomass and cellular activity of Staphylococcus by crystal violet and XTT assay, and leukocyte toxicity, measuring the increase of reactive oxygen species by chemiluminescence, and using 123-dihydrorhodamine. In addition, MC was prepared by the freeze-drying or physical mixture methods, and then characterized by scanning electron microscopy and powder X-ray diffraction. Nuclear magnetic resonance and phase solubility studies provided information at the molecular level on the structure of the MC and its association binding constants, respectively. The results obtained allowed us to conclude that MC formation is an effective pharmaceutical strategy that can reduce CP toxicity against leukocytes, while enhancing its solubility and antibiofilm activity. PMID:27516318

  19. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    Science.gov (United States)

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  20. Membrane steroid-binding protein 1 (MSBP1) negatively regulates brassinosteroid signaling by enhancing the endocytosis of BAK1

    Institute of Scientific and Technical Information of China (English)

    Li Song; Qiu-Ming Shi; Xiao-Hua Yang; Zhi-Hong Xu; Hong-Wei Xue

    2009-01-01

    Brassinosteroids (BRs) are perceived by transmembrane receptors and play vital roles in plant growth and devel-opment, as well as cell in responses to environmental stimuli. The transmembrane receptor BRI1 can directly bind to brassinolide (BL), and BAK1 interacts with BRI1 to enhance the BRll-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 (MSBP1) could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBP1 specifically interacts with the extracellular domain of BAKI in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBPI can be recovered by overexpressing BAKI or its intracellular kinase domain, sug-gesting that MSBPI may suppress BR signaling through interacting with BAK1. Subcellular localization studies re-vealed that both MSBPI and BAKI are localized to plasma membrane and endocytic vesicles and MSBPI accelerates BAKI endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAKI toward endosomes. Indeed, enhanced MSBPI expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBPI acts as a negative factor at an early step of the BR signaling pathway.

  1. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-01-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell–specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte–specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte–specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell–regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell–specific transcriptional activity. PMID:26808502

  2. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-03-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

  3. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  4. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    Science.gov (United States)

    Guo, Bihong; Li, Shaopeng; Song, Lusheng; Yang, Mo; Zhou, Wenfei; Tyagi, Deependra; Zhu, Jinsong

    2015-08-01

    A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein-protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the improvement of the protein bioassay performance, rather than the chemical changes.

  5. Southern leaf blight disease severity is correlated with decreased maize leaf epiphytic bacterial species richness and the phyllosphere bacterial diversity decline is enhanced by nitrogen fertilization

    Directory of Open Access Journals (Sweden)

    Heather eManching

    2014-08-01

    Full Text Available Plant leaves are inhabited by a diverse group of microorganisms that are important contributors to optimal growth. Biotic and abiotic effects on plant growth are usually studied in controlled settings examining response to variation in single factors and in field settings with large numbers of variables. Multi-factor experiments with combinations of stresses bridge this gap, increasing our understanding of the genotype-environment-phenotype functional map for the host plant and the affiliated epiphytic community. The maize inbred B73 was exposed to single and combination abiotic and the biotic stress treatments: low nitrogen fertilizer and high levels of infection with southern leaf blight (causal agent Cochliobolus heterostrophus. Microbial epiphyte samples were collected at the vegetative early-season phase and species composition was determined using 16S ribosomal intergenic spacer analysis. Plant traits and level of southern leaf blight disease were measured late-season. Bacterial diversity was different among stress treatment groups (P< 0.001. Lower species richness—alpha diversity--was correlated with increased severity of southern leaf blight disease when disease pressure was high. Nitrogen fertilization intensified the decline in bacterial alpha diversity. While no single bacterial ribotype was consistently associated with disease severity, small sets of ribotypes were good predictors of disease levels. Difference in leaf bacterial-epiphyte diversity early in the season were correlated with plant disease severity, supporting further tests of microbial epiphyte-disease correlations for use in predicting disease progression.

  6. Binding sites for abundant nuclear factors modulate RNA polymerase I-dependent enhancer function in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kang, J J; Yokoi, T J; Holland, M J

    1995-12-01

    The 190-base pair (bp) rDNA enhancer within the intergenic spacer sequences of Saccharomyces cerevisiae rRNA cistrons activates synthesis of the 35S-rRNA precursor about 20-fold in vivo (Mestel,, R., Yip, M., Holland, J. P., Wang, E., Kang, J., and Holland, M. J. (1989) Mol. Cell. Biol. 9, 1243-1254). We now report identification and analysis of transcriptional activities mediated by three cis-acting sites within a 90-bp portion of the rDNA enhancer designated the modulator region. In vivo, these sequences mediated termination of transcription by RNA polymerase I and potentiated the activity of the rDNA enhancer element. Two trans-acting factors, REB1 and REB2, bind independently to sites within the modulator region (Morrow, B. E., Johnson, S. P., and Warner, J. R. (1989) J. Biol. Chem. 264, 9061-9068). We show that REB2 is identical to the ABF1 protien. Site-directed mutagenesis of REB1 and ABF1 binding sites demonstrated uncoupling of RNA polymerase I-dependent termination from transcriptional activation in vivo. We conclude that REB1 and ABF1 are required for RNA polymerase I-dependent termination and enhancer function, respectively, Since REB1 and ABF1 proteins also regulate expression of class II genes and other nuclear functions, our results suggest further similarities between RNA polymerase I and II regulatory mechanisms. Two rDNA enhancers flanking a rDNA minigene stimulated RNA polymerase I transcription in a "multiplicative" fashion. Deletion mapping analysis showed that similar cis-acting sequences were required for enhancer function when positioned upstream or downstream from a rDNA minigene.

  7. Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density.

    Directory of Open Access Journals (Sweden)

    Katherine Gurdziel

    Full Text Available The Hedgehog (Hh signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A; invected (inv, which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx, the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb; and two previously untested regions of the Hh receptor-encoding patched (ptc gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.

  8. Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice

    DEFF Research Database (Denmark)

    Wu, H.; Song, Z.; Hentzer, Morten;

    2004-01-01

    Introduction: Antibiotics are used to treat bacterial infections by killing the bacteria or inhibiting their growth, but resistance to antibiotics can develop readily. The discovery that bacterial quorum-sensing regulates bacterial virulence as well as the formation of biofilms opens up new ways...... to control certain bacterial infections. Furanone compounds capable of inhibiting bacterial quorum-sensing systems have been isolated from the marine macro alga Delisea pulchra. Objectives: Two synthetic furanones were tested for their ability to attenuate bacterial virulence in the mouse models of chronic...... lung infection by targeting bacterial quorum-sensing without directly killing bacteria or inhibiting their growth. Methods: Study I. Mice with Escherichia coli MT102 [luxR-PluxI-gfp(ASV)] lung infection were injected intravenously with N-acyl homoserine lactones with or without furanones to test...

  9. Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein.

    Directory of Open Access Journals (Sweden)

    Laura A Novotny

    Full Text Available Cystic fibrosis (CF is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

  10. Biorefinery of bacterial cellulose from rice straw: enhanced enzymatic saccharification by ionic liquid pretreatment%Biorefinery of bacterial cellulose from rice straw: enhanced enzymatic saccharification by ionic liquid pretreatment

    Institute of Scientific and Technical Information of China (English)

    Hong Feng; Han Shifen

    2011-01-01

    The pretreatment of rice straw is often used to enhance the hydrolysis. 1-allyl-3-methylimidazolium chloride ( [ AMIM ] C1) is a kind of low viscous, nontoxic and recyclable ionic liquid. It was used to treat rice straw and improve the enzymatic hydrolysis of rice straw in this study. The factors influencing the pretreatment were as follows: the dosage of rice straw in [ AMIM ] Cl, crush mesh of rice straw, pretreatment temperature and time. After the pretreatment with a 3 % (the weight ratio of rice straw to ionic liquid) rice straw dosage in [AMIM]Cl at 110 ℃ for 1 h, the yield of reducing sugar of regenerated rice straw by 33 U/mL cellulase hydrolysis was 53.3 %, which was two times higher than that of un-treated rice straw (23.7 % ). More researches regarding straw biorefinery to bacterial cellulose are being performed in the lab and prospective results will be published in near future.

  11. F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice

    Science.gov (United States)

    Ozanic, Mateja; Gobin, Ivana; Brezovec, Martin; Marecic, Valentina; Trobonjaca, Zlatko; Abu Kwaik, Yousef; Santic, Marina

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia. PMID:27242974

  12. F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

    Science.gov (United States)

    Ozanic, Mateja; Gobin, Ivana; Brezovec, Martin; Marecic, Valentina; Trobonjaca, Zlatko; Abu Kwaik, Yousef; Santic, Marina

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

  13. Enhanced Expression of Aquaporin-9 in Rat Brain Edema Induced by Bacterial Lipopolysaccharides

    Institute of Scientific and Technical Information of China (English)

    Huaili WANG; Runming JIN; Peichao TIAN; Zhihong ZHUO

    2009-01-01

    To investigate the role of AQP9 in brain edema,the expression of AQP9 in an infectious rat brain edema model induced by the injection of lipopolysaccharide (LPS) was examined.Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expressions of AQP9 mRNA and protein at all observed intervals were significantly increased in LPS-treated animals in comparison with the control animals.Time-course analysis showed that the first signs of blood-brain barrier disruption and the increase of brain water content in LPS-treated animals were evident 6 h after LPS injection,with maximum value appearing at 12 h,which coincided with the expression profiles of AQP9 mRNA and protein in LPS-treated animals.The further correlation analysis revealed strong positive correlations among the brain water content,the disruption of the blood-brain barrier and the enhanced expressions of AQP9 mRNA and protein in LPS-treated animals.These results suggested that the regulation of AQP9 expression may play important roles in water movement and in brain metabolic homeostasis associated with the pathophysiology of brain edema induced by LPS injection.

  14. High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

    OpenAIRE

    Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Marco E. Bianchi; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

    1998-01-01

    We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by se...

  15. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.

    Science.gov (United States)

    Sun, Zhoutong; Lonsdale, Richard; Kong, Xu-Dong; Xu, Jian-He; Zhou, Jiahai; Reetz, Manfred T

    2015-10-12

    Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme. PMID:25891639

  16. Improved ligand binding energies derived from molecular dynamics: replicate sampling enhances the search of conformational space.

    Science.gov (United States)

    Adler, Marc; Beroza, Paul

    2013-08-26

    Does a single molecular trajectory provide an adequate sample conformational space? Our calculations indicate that for Molecular Mechanics--Poisson-Boltzmann Surface Area (MM-PBSA) measurement of protein ligand binding, a single molecular dynamics trajectory does not provide a representative sampling of phase space. For a single trajectory, the binding energy obtained by averaging over a number of molecular dynamics frames in an equilibrated system will converge after an adequate simulation time. A separate trajectory with nearly identical starting coordinates (1% randomly perturbed by 0.001 Å), however, can lead to a significantly different calculated binding energy. Thus, even though the calculated energy converges for a single molecular dynamics run, the variation across separate runs implies that a single run inadequately samples the system. The divergence in the trajectories is reflected in the individual energy components, such as the van der Waals and the electrostatics terms. These results indicate that the trajectories sample different conformations that are not in rapid exchange. Extending the length of the dynamics simulation does not resolve the energy differences observed between different trajectories. By averaging over multiple simulations, each with a nearly equivalent starting structure, we find the standard deviation in the calculated binding energy to be ∼1.3 kcal/mol. The work presented here indicates that combining MM-PBSA with multiple samples of the initial starting coordinates will produce more precise and accurate estimates of protein/ligand affinity. PMID:23845109

  17. Surface enhanced Raman optical activity as an ultra sensitive tool for ligand binding analysis

    DEFF Research Database (Denmark)

    Johannessen, Christian; Abdali, Salim

    2007-01-01

    upon azide complexation. Application of this method allows for rapid analysis of ligand binding in metalloproteins in dilute aqueous solution and could in the future, when combined with theoretical studies, increase the obtainable structural resolution of proteins beyond that of X-ray analysis....

  18. Enhanced biodegradation of alkane hydrocarbons and crude oil by mixed strains and bacterial community analysis.

    Science.gov (United States)

    Chen, Yu; Li, Chen; Zhou, Zhengxi; Wen, Jianping; You, Xueyi; Mao, Youzhi; Lu, Chunzhe; Huo, Guangxin; Jia, Xiaoqiang

    2014-04-01

    In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32 ± 4.09 to 97.41 and 87.29 ± 2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2 × 10(-3) to 28.6 × 10(-3) N/m. Based on newly found cooperation between the degrader and the coworking strain, rational investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation. PMID:24532465

  19. Overexpressing CYP71Z2 enhances resistance to bacterial blight by suppressing auxin biosynthesis in rice.

    Directory of Open Access Journals (Sweden)

    Wenqi Li

    Full Text Available The hormone auxin plays an important role not only in the growth and development of rice, but also in its defense responses. We've previously shown that the P450 gene CYP71Z2 enhances disease resistance to pathogens through regulation of phytoalexin biosynthesis in rice, though it remains unclear if auxin is involved in this process or not.The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation.These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided.

  20. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Bihong [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Li, Shaopeng [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Department of Chemistry, Tsinghua University, Beijing 100084 (China); Song, Lusheng [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Yang, Mo; Zhou, Wenfei; Tyagi, Deependra [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); University of Chinese Academy of Sciences, Yuquan Rd., 19(A), Beijing 100049 (China); Zhu, Jinsong, E-mail: jizhu88@gmail.com [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China)

    2015-08-01

    Highlights: • A simple and robust plasma-treated ultrathin polystyrene film surface was developed for protein biosensing. • The surface was optimized by evaluating up to 120 types of fabrication parameters with high-throughput analytical methods. • The optimized surface showed a 620% improvement of the protein detection signal and 210% protein binding per immobilized protein ligand compared with a self-assembled monolayer surface. - Abstract: A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein–protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the

  1. A Manganese(V)-Oxo Complex: Synthesis by Dioxygen Activation and Enhancement of Its Oxidizing Power by Binding Scandium Ion.

    Science.gov (United States)

    Hong, Seungwoo; Lee, Yong-Min; Sankaralingam, Muniyandi; Vardhaman, Anil Kumar; Park, Young Jun; Cho, Kyung-Bin; Ogura, Takashi; Sarangi, Ritimukta; Fukuzumi, Shunichi; Nam, Wonwoo

    2016-07-13

    A mononuclear non-heme manganese(V)-oxo complex, [Mn(V)(O)(TAML)](-) (1), was synthesized by activating dioxygen in the presence of olefins with weak allylic C-H bonds and characterized structurally and spectroscopically. In mechanistic studies, the formation rate of 1 was found to depend on the allylic C-H bond dissociation energies (BDEs) of olefins, and a kinetic isotope effect (KIE) value of 16 was obtained in the reactions of cyclohexene and cyclohexene-d10. These results suggest that a hydrogen atom abstraction from the allylic C-H bonds of olefins by a putative Mn(IV)-superoxo species, which is formed by binding O2 by a high-spin (S = 2) [Mn(III)(TAML)](-) complex, is the rate-determining step. A Mn(V)-oxo complex binding Sc(3+) ion, [Mn(V)(O)(TAML)](-)-(Sc(3+)) (2), was also synthesized in the reaction of 1 with Sc(3+) ion and then characterized using various spectroscopic techniques. The binding site of the Sc(3+) ion was proposed to be the TAML ligand, not the Mn-O moiety, probably due to the low basicity of the oxo group compared to the basicity of the amide carbonyl group in the TAML ligand. Reactivity studies of the Mn(V)-oxo intermediates, 1 and 2, in oxygen atom transfer and electron-transfer reactions revealed that the binding of Sc(3+) ion at the TAML ligand of Mn(V)-oxo enhanced its oxidizing power with a positively shifted one-electron reduction potential (ΔEred = 0.70 V). This study reports the first example of tuning the second coordination sphere of high-valent metal-oxo species by binding a redox-inactive metal ion at the supporting ligand site, thereby modulating their electron-transfer properties as well as their reactivities in oxidation reactions. PMID:27310336

  2. A Manganese(V)-Oxo Complex: Synthesis by Dioxygen Activation and Enhancement of Its Oxidizing Power by Binding Scandium Ion.

    Science.gov (United States)

    Hong, Seungwoo; Lee, Yong-Min; Sankaralingam, Muniyandi; Vardhaman, Anil Kumar; Park, Young Jun; Cho, Kyung-Bin; Ogura, Takashi; Sarangi, Ritimukta; Fukuzumi, Shunichi; Nam, Wonwoo

    2016-07-13

    A mononuclear non-heme manganese(V)-oxo complex, [Mn(V)(O)(TAML)](-) (1), was synthesized by activating dioxygen in the presence of olefins with weak allylic C-H bonds and characterized structurally and spectroscopically. In mechanistic studies, the formation rate of 1 was found to depend on the allylic C-H bond dissociation energies (BDEs) of olefins, and a kinetic isotope effect (KIE) value of 16 was obtained in the reactions of cyclohexene and cyclohexene-d10. These results suggest that a hydrogen atom abstraction from the allylic C-H bonds of olefins by a putative Mn(IV)-superoxo species, which is formed by binding O2 by a high-spin (S = 2) [Mn(III)(TAML)](-) complex, is the rate-determining step. A Mn(V)-oxo complex binding Sc(3+) ion, [Mn(V)(O)(TAML)](-)-(Sc(3+)) (2), was also synthesized in the reaction of 1 with Sc(3+) ion and then characterized using various spectroscopic techniques. The binding site of the Sc(3+) ion was proposed to be the TAML ligand, not the Mn-O moiety, probably due to the low basicity of the oxo group compared to the basicity of the amide carbonyl group in the TAML ligand. Reactivity studies of the Mn(V)-oxo intermediates, 1 and 2, in oxygen atom transfer and electron-transfer reactions revealed that the binding of Sc(3+) ion at the TAML ligand of Mn(V)-oxo enhanced its oxidizing power with a positively shifted one-electron reduction potential (ΔEred = 0.70 V). This study reports the first example of tuning the second coordination sphere of high-valent metal-oxo species by binding a redox-inactive metal ion at the supporting ligand site, thereby modulating their electron-transfer properties as well as their reactivities in oxidation reactions.

  3. X-ray Crystal Structure of the Novel Enhanced-Affinity Glucocorticoid Agonist Fluticasone Furoate in the Glucocorticoid Receptor−Ligand Binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Biggadike, Keith; Bledsoe, Randy K.; Hassell, Anne M.; Kirk, Barrie E.; McLay, Iain M.; Shewchuk, Lisa M.; Stewart, Eugene L. (GSKNC); (GSK)

    2008-07-08

    An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17{alpha} furoate ester to occupy more fully the lipophilic 17{alpha} pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.

  4. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

    Science.gov (United States)

    Feo, S; Antona, V; Barbieri, G; Passantino, R; Calì, L; Giallongo, A

    1995-01-01

    To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells. PMID:7565752

  5. Rhizospheric Bacterial Strain Brevibacterium casei MH8a Colonizes Plant Tissues and Enhances Cd, Zn, Cu Phytoextraction by White Mustard.

    Science.gov (United States)

    Płociniczak, Tomasz; Sinkkonen, Aki; Romantschuk, Martin; Sułowicz, Sławomir; Piotrowska-Seget, Zofia

    2016-01-01

    Environmental pollution by heavy metals has become a serious problem in the world. Phytoextraction, which is one of the plant-based technologies, has attracted the most attention for the bioremediation of soils polluted with these contaminants. The aim of this study was to determine whether the multiple-tolerant bacterium, Brevibacterium casei MH8a isolated from the heavy metal-contaminated rhizosphere soil of Sinapis alba L., is able to promote plant growth and enhance Cd, Zn, and Cu uptake by white mustard under laboratory conditions. Additionally, the ability of the rifampicin-resistant spontaneous mutant of MH8a to colonize plant tissues and its mechanisms of plant growth promotion were also examined. In order to assess the ecological consequences of bioaugmentation on autochthonous bacteria, the phospholipid fatty acid (PLFA) analysis was used. The MH8a strain exhibited the ability to produce ammonia, 1-amino-cyclopropane-1-carboxylic acid deaminase, indole 3-acetic acid and HCN but was not able to solubilize inorganic phosphate and produce siderophores. Introduction of MH8a into soil significantly increased S. alba biomass and the accumulation of Cd (208%), Zn (86%), and Cu (39%) in plant shoots in comparison with those grown in non-inoculated soil. Introduced into the soil, MH8a was able to enter the plant and was found in the roots and leaves of inoculated plants thus indicating its endophytic features. PLFA analysis revealed that the MH8a that was introduced into soil had a temporary influence on the structure of the autochthonous bacterial communities. The plant growth-promoting features of the MH8a strain and its ability to enhance the metal uptake by white mustard and its long-term survival in soil as well as its temporary impact on autochthonous microorganisms make the strain a suitable candidate for the promotion of plant growth and the efficiency of phytoextraction. PMID:26909087

  6. Rhizospheric bacterial strain Brevibacterium casei MH8a colonizes plant tissues and enhances Cd, Zn, Cu phytoextraction by white mustard

    Directory of Open Access Journals (Sweden)

    Tomasz ePłociniczak

    2016-02-01

    Full Text Available Environmental pollution by heavy metals has become a serious problem in the world. Phytoextraction, which is one of the plant-based technologies, has attracted the most attention for the bioremediation of soils polluted with these contaminants.The aim of this study was to determine whether the multiple-tolerant bacterium, Brevibacterium casei MH8a isolated from the heavy metal-contaminated rhizosphere soil of Sinapis alba L., is able to promote plant growth and enhance Cd, Zn and Cu uptake by white mustard under laboratory conditions. Additionally, the ability of the rifampicin-resistant spontaneous mutant of MH8a to colonize plant tissues and its mechanisms of plant growth promotion were also examined. In order to assess the ecological consequences of bioaugmentation on autochthonous bacteria, the phospholipid fatty acid (PLFA analysis was used. The MH8a strain exhibited the ability to produce ammonia, 1-amino-cyclopropane-1-carboxylic acid deaminase, indole 3-acetic acid and HCN but was not able to solubilize inorganic phosphate and produce siderophores. Introduction of MH8a into soil significantly increased S. alba biomass and the accumulation of Cd (208%, Zn (86% and Cu (39% in plant shoots in comparison with those grown in non-inoculated soil. Introduced into the soil, MH8a was able to enter the plant and was found in the roots and leaves of inoculated plants thus indicating its endophytic features. PLFA analysis revealed that the MH8a that was introduced into soil had a temporary influence on the structure of the autochthonous bacterial communities. The plant growth-promoting features of the MH8a strain and its ability to enhance the metal uptake by white mustard and its long-term survival in soil as well as its temporary impact on autochthonous microorganisms make the strain a suitable candidate for the promotion of plant growth and the efficiency of phytoextraction.

  7. Surface modification of bacterial cellulose nanofibers for property enhancement of optically transparent composites: dependence on acetyl-group DS.

    Science.gov (United States)

    Ifuku, Shinsuke; Nogi, Masaya; Abe, Kentaro; Handa, Keishin; Nakatsubo, Fumiaki; Yano, Hiroyuki

    2007-06-01

    Bacterial cellulose (BC) nanofibers were acetylated to enhance the properties of optically transparent composites of acrylic resin reinforced with the nanofibers. A series of BC nanofibers acetylated from degree-of-substitution (DS) 0 to 1.76 were obtained. X-ray diffraction profiles indicated that acetylation proceeded from the surface to the core of BC nanofibers, and scanning electron microscopy images showed that the volume of nanofibers increases by the bulky acetyl group. Since acetylation decreased the refractive index of cellulose, regular transmittance of composites comprised of 63% BC nanofiber was improved, and deterioration at 580 nm because of fiber reinforcement was suppressed to only 3.4%. Acetylation of nanofibers changed their surface properties and reduced the moisture content of the composite to about one-third that of untreated composite, although excessive acetylation increased hygroscopicity. Furthermore, acetylation was found to reduce the coefficient of thermal expansion of a BC sheet from 3 x 10(-6) to below 1 x 10(-6) 1/K.

  8. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    OpenAIRE

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-af...

  9. Biofunctionalization of titanium surfaces with heparin-binding biomolecules to enhance osteointegration of implants

    OpenAIRE

    Garrido Domínguez, Beatriz

    2015-01-01

    For orthopaedic, dental or craniofacial applications osteointegration is critical for short-term initial stability and long-term success of the implant. Important efforts have been made in the past to optimize the osteointegration of titanium implants in bone-contact applications, focusing mainly on biofunctionalization methods of its surface to reduce healing times and accelerate integration into the host tissue. In the present in vitro study a heparin binding peptide (FHRRIKA) and a r...

  10. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    Science.gov (United States)

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function. PMID:27131598

  11. Conjugation of benzylvanillin and benzimidazole structure improves DNA binding with enhanced antileukemic properties.

    Directory of Open Access Journals (Sweden)

    Zena A Al-Mudaris

    Full Text Available Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy-3-methoxybenzaldehyde compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl-2-(2-benzyloxy-3-methoxyphenyl-1H-benzimidazole, 2XP, and (R and (S-1-(2-benzyloxy-3-methoxyphenyl-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1, the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1 has weak anticancer activity even after it was combined with the benzimidazole (2MP, but after addition of another benzylvanillin structure (2XP, stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS significantly improved

  12. Biodistribution of a 67Ga-labeled anti-TNF VHH single-domain antibody containing a bacterial albumin-binding domain (Zag)

    International Nuclear Information System (INIS)

    Introduction: Small domain antibodies (sdAbs) present high potential for both molecular in vivo imaging and therapy. Owing to the low molecular weight they are rapidly cleared from blood circulation, and new strategies to extend their half-lifes are needed for therapeutic applications. We have selected a bacterial albumin-binding domain (ABD) from protein Zag to be fused to an anti-tumor necrosis factor (TNF) single variable-domain heavy-chain region antibody (VHH) to delay blood clearance, and evaluated the biodistribution profile of the fusion protein. Methods: The anti-TNF VHH and the fusion protein VHH-Zag were conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). The anti-TNF and albumin-binding properties of the conjugates NOTA-VHH and NOTA-VHH-Zag were assessed by enzyme-linked immunosorbent assay (ELISA). The radioconjugates 67Ga-NOTA-VHH and 67Ga-NOTA-VHH-Zag were obtained by reaction of 67GaCl3 with the corresponding conjugates at room temperature. Biodistribution studies were performed in healthy female CD-1 mice. Results: The immunoreactivity of the VHH-based proteins is preserved upon conjugation to NOTA as well as after radiometallation. The radiochemical purity of the radioconjugates was higher than 95% as determined by ITLC-SG after purification by gel filtration. The biodistribution studies showed that the Zag domain affected the pharmacokinetic properties of VHH, with impressive differences in blood clearance (0.028 ± 0.004 vs 1.7 ± 0.8 % I.A./g) and total excretion (97.8 ± 0.6 vs 25.5 ± 2.1 % I.A.) for 67Ga-NOTA-VHH and 67Ga-NOTA-VHH-Zag, respectively, at 24 h p.i. Conclusion: The Zag domain prolonged the circulation time of VHH by reducing the blood clearance of the labeled fusion protein 67Ga-NOTA-VHH-Zag. In this way, the anti-TNF VHH in fusion with the Zag ABD presents a higher therapeutic potential than the unmodified VHH

  13. The DUF582 Proteins of Chlamydia trachomatis Bind to Components of the ESCRT Machinery, Which Is Dispensable for Bacterial Growth In vitro

    Science.gov (United States)

    Vromman, François; Perrinet, Stéphanie; Gehre, Lena; Subtil, Agathe

    2016-01-01

    Chlamydiae are Gram negative bacteria that develop exclusively inside eukaryotic host cells, within a membrane-bounded compartment. Members of the family Chlamydiaceae, such as Chlamydia trachomatis, are pathogenic species infecting vertebrates. They have a very reduced genome and exploit the capacities of their host for their own development, mainly through the secretion of proteins tailored to interfere with eukaryotic processes, called effector proteins. All Chlamydiaceae possess genes coding for four to five effectors that share a domain of unknown function (DUF582). Here we show that four of these effectors, which represent the conserved set in all Chlamydiaceae, accumulate in the infectious form of C. trachomatis, and are therefore likely involved in an early step of the developmental cycle. The fifth member of the family, CT621, is specific to C. trachomatis, and is secreted during the growth phase. Using a two-hybrid screen in yeast we identified an interaction between the host protein Hrs and the DUF582, which we confirmed by co-immunoprecipitations in co-transfected mammalian cells. Furthermore, we provide biochemical evidence that a second domain of one of the DUF582 proteins, CT619, binds the host protein Tsg101. Hrs and Tsg101 are both implicated in a well conserved machinery of the eukaryotic cell called the ESCRT machinery, which is involved in several cellular processes requiring membrane constriction. Using RNA interference targeting proteins implicated at different stages of ESCRT-driven processes, or inhibition by expression of a dominant negative mutant of VPS4, we demonstrated that this machinery was dispensable for bacterial entry, multiplication and differentiation into infectious progeny, and for uptake of glycogen into the parasitophorous vacuole. In light of these observations we discuss how the DUF582 proteins might target the ESCRT machinery during infection. PMID:27774439

  14. Expedient chemical synthesis of 75mer DNA binding domain of MafA: an insight on its binding to insulin enhancer.

    Science.gov (United States)

    Pellegrino, Sara; Annoni, Chiara; Contini, Alessandro; Clerici, Francesca; Gelmi, Maria Luisa

    2012-11-01

    An expedient chemical synthesis of a 75mer peptide corresponding to the DNA binding domain (DBD, 227-301) of the human MafA leucine zipper transcription factor is reported. The application of microwave-assisted solid phase peptide synthesis (MW-SPPS) with a protocol modified respect to the standard one allowed obtaining the desired 75mer peptide in a short time with high quantity and optimal purity. MW-SPPS methodology was thus demonstrated as a valuable alternative to recombinant methods to obtain protein domains. Considering that recent findings suggest an involvement of MafA in the pathogenesis of diabetes mellitus, we also performed circular dichroism studies both on DBD folding and its interaction with MafA recognition element (MARE) on insulin enhancer. From our results, it was evicted that a disorder to order transition occurs after DBD interaction with insulin MARE which is mediated by specific structural elements on the N-terminus of the DBD. PMID:22476346

  15. Liver Fatty Acid Binding Protein Gene Ablation Enhances Age-Dependent Weight Gain in Male Mice

    OpenAIRE

    Martin, Gregory G.; Atshaves, Barbara P.; McIntosh, Avery L.; Payne, H. Ross; Mackie, John T.; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 mo. While young (2-3 mo) L-FABP null mice displayed no visually obvi...

  16. AMF-1/Gps2 Binds p300 and Enhances Its Interaction with Papillomavirus E2 Proteins

    OpenAIRE

    Peng, Yu-Cai; Breiding, David E.; Sverdrup, Francis; Richard, James; Androphy, Elliot J.

    2000-01-01

    The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208–7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 an...

  17. The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin.

    Science.gov (United States)

    Gao, Feng; Gulay, Suna P; Kasprzak, Wojciech; Dinman, Jonathan D; Shapiro, Bruce A; Simon, Anne E

    2013-11-01

    The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation. PMID:23986599

  18. Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

    Directory of Open Access Journals (Sweden)

    Hastie CJ

    2006-04-01

    Full Text Available Abstract Background Glycogen Synthase Kinase-3 (GSK3 activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK, Glucose-6 Phosphatase (G6Pase and IGF binding protein-1 (IGFBP1 gene expression. CAAT/enhancer binding protein alpha (C/EBPα regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226 by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters. Results C/EBPα represses the IGFBP1 thymine-rich insulin response element (TIRE, but mutation of T222 or T226 of C/EBPα to non-phosphorylatable alanines has no effect on C/EBPα activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence. Phosphorylation of T222/T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBPα activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/or S230 to alanine residues. Finally, we demonstrate that C/EBPα is a very poor substrate for GSK3 in vitro and in cells. Conclusion The work demonstrates an important role for this domain in the regulation of C/EBPα activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBPα activity is regulated by direct phosphorylation by GSK3.

  19. Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

    OpenAIRE

    Chu, Ming; Ding, Ran; Chu, Zheng-yun; Zhang, Ming-bo; Liu, Xiao-Yan; Xie, Shao-Hua; Zhai, Yan-jun; Wang, Yue-dan

    2014-01-01

    Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanis...

  20. Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

    OpenAIRE

    Mouhssin Oufir; Bisset, Leslie R.; Hoffmann, Stefan R. K.; Gongda Xue; Stephan Klauser; Bianca Bergamaschi; Alain Gervaix; Jürg Böni; Jörg Schüpbach; Bernd Gutte

    2011-01-01

    An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 r...

  1. Enhancing the Decolorizing and Degradation Ability of Bacterial Consortium Isolated from Textile Effluent Affected Area and Its Application on Seed Germination

    OpenAIRE

    2015-01-01

    A bacterial consortium BMP1/SDSC/01 consisting of six isolates was isolated from textile effected soil, sludge, and textile effluent from Hudiara drain near Nishat Mills Limited, Ferozepur Road, Lahore, Pakistan. It was selected because of being capable of degrading and detoxifying red, green, black, and yellow textile dyes. The pH and supplements were optimized to enhance the decolorization ability of the selected consortium. The results indicated that decolorizing ability of consortium for ...

  2. Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease.

    Science.gov (United States)

    Hernandez, Ciria C; Nascimento, Jose H; Chaves, Elen A; Costa, Patricia C; Masuda, Masako O; Kurtenbach, Eleonora; Campos DE Carvalho, Antonio C; Gimenez, Luis E

    2008-01-01

    Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

  3. Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas’ Disease

    Science.gov (United States)

    Hernández, Ciria C.; Nascimento, José H.; Chaves, Elen A.; Costa, Patrícia C.; Masuda, Masako O.; Kurtenbach, Eleonora; Campos de Carvalho, Antônio C.; Giménez, Luis E.

    2009-01-01

    Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M2-muscarinic acetylcholine receptors (M2AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M2AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M2AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [3H]-N-methyl scopolamine ([3H]-NMS) in allosterism binding assays. A peptide corresponding to the M2AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [3H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [3H]-NMS dissociation right shifted from an IC50 of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10−8, 1.33 × 10−7, and 2.0 × 10−7 mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M2AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

  4. Human Intersectin 2 (ITSN2 binds to Eps8 protein and enhances its degradation

    Directory of Open Access Journals (Sweden)

    Xiaofeng Ding

    2012-03-01

    Full Text Available Participates in actin remodeling through Rac and receptor endocytosisvia Rab5. Here, we used yeast two-hybrid systemwith Eps8 as bait to screen a human brain cDNA library.ITSN2 was identified as the novel binding factor of Eps8. Theinteraction between ITSN2 and Eps8 was demonstrated by thein vivo co-immunoprecipitation and colocalization assays andthe in vitro GST pull-down assays. Furthermore, we mappedthe interaction domains to the region between amino acids260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition,protein stability assays and immunofluorescence analysisshowed ITSN2 overexpression induced the degradation ofEps8 proteins, which was markedly alleviated with the lysosomeinhibitor NH4Cl treatment. Taken together, our resultssuggested ITSN2 interacts with Eps8 and stimulates the degradationof Eps8 proteins. [BMB reports 2012; 45(3: 183-188

  5. Introduction of D-Phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides

    OpenAIRE

    Lu, Jie; Hathaway, Helen J.; Royce, Melanie E.; Prossnitz, Eric R.; Miao, Yubin

    2014-01-01

    The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys6-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys6-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys6 to generate new DOTA-D-Phe-Ahx-(D-Lys6-GnRH) or DOTA-Ahx-D-Phe-(D-Lys6-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys6-GnRH1) (36.1 nM), the introd...

  6. STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.

    Science.gov (United States)

    Zhao, Guiling; Li, Tianyu; Brochet, Didier X P; Rosenberg, Paul B; Lederer, W J

    2015-08-25

    In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells. PMID:26261328

  7. Resistance Induction and Enhanced Tuber Production by Pre-inoculation with Bacterial Strains in Potato Plants against Phytophthora infestans

    OpenAIRE

    Kim, Hyo-Jeong; Jeun, Yong-Chull

    2006-01-01

    Efficacy of resistance induction by the bacterial isolates Pseudomonas putida (TRL2-3), Micrococcus luteus (TRK2-2) and Flexibacteraceae bacterium (MRL412), which were isolated from the rhizosphere of plants growing in Jeju Mountain, were tested in a greenhouse. The disease severity caused by Phytophthora infestans was effectively reduced in the potato plants pre-inoculated with bacterial isolates compared with those of the untreated control plants growing in a greenhouse. In order to estimat...

  8. The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2 upregulates galectin-7 expression in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Carole G Campion

    Full Text Available Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2, the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

  9. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    Science.gov (United States)

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-01

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  10. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  11. Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

    Science.gov (United States)

    Futaki, S; Ueno, H; Martinez del Pozo, A; Pospischil, M A; Manning, J M; Ringe, D; Stoddard, B; Tanizawa, K; Yoshimura, T; Soda, K

    1990-12-25

    In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild

  12. Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-. kappa. B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, K.; Scheidereit, C.; Roeder, R.G. (Rockefeller Univ., New York, NY (USA))

    1988-07-01

    The enhancer-binding factor NF-{kappa}B, which is found only in cells that transcribe immunoglobulin light chain genes, has been purified from nuclear extracts of Namalwa cells (human Burkitt lymphoma cells) by sequence-specific DNA affinity chromatography. The purified NF-{kappa}B has been identified as a 51-kDa polypeptide by UV-crosslinking analysis. Footprint and methylation-interference analyses have shown that purified NF-{kappa}B has a binding activity specific for the {kappa} light chain enhancer sequence. The purified factor activated in vitro transcription of the human immunodeficiency virus type I promoter by binding to an upstream NF-{kappa}B-binding site.

  13. CNS depressants accelerate the dissociation of /sup 35/S-TBPS binding and GABA enhances their displacing potencies

    Energy Technology Data Exchange (ETDEWEB)

    Maksay, G.; Ticku, M.K.

    1988-01-01

    The specific binding of /sup 35/S-t-butylbicyclophosphorothionate (TBPS) was studied in synaptosomal membranes of rat cerebral cortex. The displacing potencies of eleven CNS depressants and three convulsants were determined in the presence of 1 /sup +/M GABA and 10 nM R 5135. GABA enhanced the displacing potencies of depressants of most diverse chemical structures: diaryltriazine (LY 81067), pyrazolopyridine (etazolate), cinnamide, glutarimide, 2,3-benzodiazepine (tofizopam) and alcohol derivatives, barbiturates, (+)etomidate, methaqualone and meprobamate. In contrast, the IC/sub 50/ values of convulsants (picrotoxinin, pentetrazol and the barbiturate enantiomer S(+)MPPB) were not significantly affected. The depressants accelerated either basal or GABA-augmented dissociation of /sup 35/-TBPS mainly by increasing the contribution of its rapid first phase.

  14. Human α-Defensin Expression Is Not Dependent on CCAAT/Enhancer Binding Protein-ε in a Murine Model

    DEFF Research Database (Denmark)

    Glenthøj, Andreas; Dahl, Sara; Larsen, Maria T;

    2014-01-01

    Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-ε (C/EBP-ε) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore......, azurophil granules contain diminished amounts of their most abundant proteins, α-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-ε. Since mice do not express myeloid defensins, they cannot per se be used...... by lack of C/EBP-ε in these mice. Transduction of C/EBP-ε into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern...

  15. Evaluation of cell binding peptide (p15) with silk fibre enhanced hydroxyappatite bone substitute for posterolateral spinal fusion in sheep

    DEFF Research Database (Denmark)

    Axelsen, M.; Jespersen, Stig; Overgaard, Søren;

    2015-01-01

    Background: Spinal fusion is indicated in the surgical management of various spinal disorders. To ensure stabile fusion, bone graft materials are essential. Traditionally allo- or autograft has been used, but both are associated with limitations. Synthetic bone graft materials that reassemble today...... on the surface of bone forming cells. The binding initiates natural intra- and extracellular signalling pathways, inducing production of growth factors, bone morphogenic proteins and cytokines. P15 peptide has previously shown to improve osteoinductive properties when coated on graft materials. Purpose...... two level uninstrumented PLF at level L2/L3 and L4/L5. Levels were randomised to receive silk fibre enhanced ABM graft with or graft without P15 coating. The sheep were sacrificed after 4.5 months. Levels were harvested and evaluated with Micro-CT 50 scanner and qualitative histology. Fusion rates...

  16. Optimized Gamma Synchronization Enhances Functional Binding of Fronto-Parietal Cortices in Mathematically Gifted Adolescents during Deductive Reasoning.

    Science.gov (United States)

    Zhang, Li; Gan, John Q; Wang, Haixian

    2014-01-01

    As enhanced fronto-parietal network has been suggested to support reasoning ability of math-gifted adolescents, the main goal of this EEG source analysis is to investigate the temporal binding of the gamma-band (30-60 Hz) synchronization between frontal and parietal cortices in adolescents with exceptional mathematical ability, including the functional connectivity of gamma neurocognitive network, the temporal dynamics of fronto-parietal network (phase-locking durations and network lability in time domain), and the self-organized criticality of synchronizing oscillation. Compared with the average-ability subjects, the math-gifted adolescents show a highly integrated fronto-parietal network due to distant gamma phase-locking oscillations, which is indicated by lower modularity of the global network topology, more "connector bridges" between the frontal and parietal cortices and less "connector hubs" in the sensorimotor cortex. The time domain analysis finds that, while maintaining more stable phase dynamics of the fronto-parietal coupling, the math-gifted adolescents are characterized by more extensive fronto-parietal connection reconfiguration. The results from sample fitting in the power-law model further find that the phase-locking durations in the math-gifted brain abides by a wider interval of the power-law distribution. This phase-lock distribution mechanism could represent a relatively optimized pattern for the functional binding of frontal-parietal network, which underlies stable fronto-parietal connectivity and increases flexibility of timely network reconfiguration. PMID:24966829

  17. Albumin-based nanoparticle trehalose lyophilisation stress-down to preserve structure/function and enhanced binding.

    Science.gov (United States)

    Siri, Macarena; Grasselli, Mariano; Alonso, Silvia Del V

    2016-07-15

    The aim of this study was to preserve albumin nanoparticle structure/function during the lyophilisation process. Bovine serum albumin nanoparticles were obtained by γ-irradiation. Nanoparticles were lyophilised in buffer, miliQ water or in trehalose/miliQ solution. The size and charge of the nanoparticles were tested after lyophilisation by light scattering and Z potential. The most relevant results in size of BSA nanoparticle were those lyophilised in PBS between 20 and 350nm, assembled in different aggregates, and negative Z potential obtained was 37±8mV in all, and those nanoparticles lyophilised with trehalose had a size range of 70±2nm and a negative Z potential of 20±5mV. Structure determination of surface aminoacids SH groups in the BSA NP lyophilised in PBS showed an increase in the free SH groups. Different aggregates had different amount of SH groups exposure from 55 to 938 (from smaller to bigger aggregates), whereas BSA NP lyophilised with trehalose showed no significant difference if compared with BSA NP. The binding properties of the BSA nanoparticle with a theragnostic probe (merocyanine 540) were studied after lyophilisation. Results showed more affinity between the BSA NP lyophilised with trehalose than that observed with non lyophilised BSA NP. As a result, the lyophilisation condition in trehalose 100μM solution is the best one to preserve the BSA NP structure/function and the one with the enhance binding affinity of the BSA NP. PMID:27174378

  18. Optimized gamma synchronization enhances functional binding of fronto-parietal cortices in mathematically gifted adolescents during deductive reasoning

    Directory of Open Access Journals (Sweden)

    Li eZhang

    2014-06-01

    Full Text Available As enhanced fronto-parietal network has been suggested to support reasoning ability of math-gifted adolescents, the main goal of this EEG source analysis is to investigate the temporal binding of the gamma-band (30-60Hz synchronization between frontal and parietal cortices in adolescents with exceptional mathematical ability, including the functional connectivity of gamma neurocognitive network, the temporal dynamics of fronto-parietal network (phase-locking durations and network lability in time domain, and the self-organized criticality of synchronizing oscillation. Compared with the average-ability subjects, the math-gifted adolescents show a highly integrated fronto-parietal network due to distant gamma phase-locking oscillations, which is indicated by lower modularity of the global network topology, more connector bridges between the frontal and parietal cortices and less connector hubs in the sensorimotor cortex. The time-domain analysis finds that, while maintaining more stable phase dynamics of the fronto-parietal coupling, the math-gifted adolescents are characterized by more extensive fronto-parietal connection reconfiguration. The results from sample fitting in the power-law model further find that the phase-locking durations in the math-gifted brain abides by a wider interval of the power-law distribution. This phase-lock distribution mechanism could represent a relatively optimized pattern for the functional binding of frontal-parietal network, which underlies stable fronto-parietal connectivity and increases flexibility of timely network reconfiguration.

  19. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    International Nuclear Information System (INIS)

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  20. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    Energy Technology Data Exchange (ETDEWEB)

    Magno, Aaron L. [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ingley, Evan [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Brown, Suzanne J. [Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Conigrave, Arthur D. [School of Molecular Bioscience, University of Sydney, New South Wales 2000 (Australia); Ratajczak, Thomas [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ward, Bryan K., E-mail: bryanw@cyllene.uwa.edu.au [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia)

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  1. Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax.

    Science.gov (United States)

    Bajpai, R; Matulis, S M; Wei, C; Nooka, A K; Von Hollen, H E; Lonial, S; Boise, L H; Shanmugam, M

    2016-07-28

    Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM. PMID:26640142

  2. X-Aptamers: A bead-based selection method for random incorporation of drug-like moieties onto next-generation aptamers for enhanced binding

    OpenAIRE

    He, WeiGuo; Elizondo-Riojas, Miguel-Angel; LI, XIN; Lokesh, Ganesh Lakshmana Rao; Somasunderam, Anoma; Thiviyanathan, Varatharasa; Volk, David E.; Durland, Ross H.; Englehardt, Johnnie; Cavasotto, Claudio N.; Gorenstein, David G.

    2012-01-01

    By combining pseudo-random bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities to the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) demonstrated to bind to CD44-HABD, to a complete monothioate backbone substituted aptamer to increase its binding affini...

  3. Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.

    OpenAIRE

    Frattini, M G; Laimins, L A

    1994-01-01

    The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by ...

  4. Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

    DEFF Research Database (Denmark)

    Burmølle, Mette; Webb, J.S.; Rao, D.;

    2006-01-01

    Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated...... specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.......Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated......-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less...

  5. Deficiency of thioredoxin binding protein-2 (TBP-2 enhances TGF-β signaling and promotes epithelial to mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    So Masaki

    Full Text Available BACKGROUND: Transforming growth factor beta (TGF-β has critical roles in regulating cell growth, differentiation, apoptosis, invasion and epithelial-mesenchymal transition (EMT of various cancer cells. TGF-β-induced EMT is an important step during carcinoma progression to invasion state. Thioredoxin binding protein-2 (TBP-2, also called Txnip or VDUP1 is downregulated in various types of human cancer, and its deficiency results in the earlier onset of cancer. However, it remains unclear how TBP-2 suppresses the invasion and metastasis of cancer. PRINCIPAL FINDINGS: In this study, we demonstrated that TBP-2 deficiency increases the transcriptional activity in response to TGF-β and also enhances TGF-β-induced Smad2 phosphorylation levels. Knockdown of TBP-2 augmented the TGF-β-responsive expression of Snail and Slug, transcriptional factors related to TGF-β-mediated induction of EMT, and promoted TGF-β-induced spindle-like morphology consistent with the depletion of E-Cadherin in A549 cells. CONCLUSIONS/SIGNIFICANCE: Our results indicate that TBP-2 deficiency enhances TGF-β signaling and promotes TGF-β-induced EMT. The control of TGF-β-induced EMT is critical for the inhibition of the invasion and metastasis. Thus TBP-2, as a novel regulatory molecule of TGF-β signaling, is likely to be a prognostic indicator or a potential therapeutic target for preventing tumor progression.

  6. EDTA addition enhances bacterial respiration activities and hydrocarbon degradation in bioaugmented and non-bioaugmented oil-contaminated desert soils.

    Science.gov (United States)

    Al Kharusi, Samiha; Abed, Raeid M M; Dobretsov, Sergey

    2016-03-01

    The low number and activity of hydrocarbon-degrading bacteria and the low solubility and availability of hydrocarbons hamper bioremediation of oil-contaminated soils in arid deserts, thus bioremediation treatments that circumvent these limitations are required. We tested the effect of Ethylenediaminetetraacetic acid (EDTA) addition, at different concentrations (i.e. 0.1, 1 and 10 mM), on bacterial respiration and biodegradation of Arabian light oil in bioaugmented (i.e. with the addition of exogenous alkane-degrading consortium) and non-bioaugmented oil-contaminated desert soils. Post-treatment shifts in the soils' bacterial community structure were monitored using MiSeq sequencing. Bacterial respiration, indicated by the amount of evolved CO2, was highest at 10 mM EDTA in bioaugmented and non-bioaugmented soils, reaching an amount of 2.2 ± 0.08 and 1.6 ± 0.02 mg-CO2 g(-1) after 14 days of incubation, respectively. GC-MS revealed that 91.5% of the C14-C30 alkanes were degraded after 42 days when 10 mM EDTA and the bacterial consortium were added together. MiSeq sequencing showed that 78-91% of retrieved sequences in the original soil belonged to Deinococci, Alphaproteobacteria, Gammaproteobacteia and Bacilli. The same bacterial classes were detected in the 10 mM EDTA-treated soils, however with slight differences in their relative abundances. In the bioaugmented soils, only Alcanivorax sp. MH3 and Parvibaculum sp. MH21 from the exogenous bacterial consortium could survive until the end of the experiment. We conclude that the addition of EDTA at appropriate concentrations could facilitate biodegradation processes by increasing hydrocarbon availability to microbes. The addition of exogenous oil-degrading bacteria along with EDTA could serve as an ideal solution for the decontamination of oil-contaminated desert soils.

  7. Influenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances susceptibility to secondary MRSA infection

    Science.gov (United States)

    Sun, Keer; Metzger, Dennis W.

    2014-01-01

    Methicillin-resistant S. aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. Here we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, despite the fact that MRSA super-infection had no significant effect on viral burdens. Compared to mice infected with bacteria alone, post-influenza MRSA infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species (ROS) levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for ROS production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91phox-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to WT animals, gp91phox−/− mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91phox+/− mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection. PMID:24563256

  8. Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability

    NARCIS (Netherlands)

    Swainsbury, David J K; Scheidelaar, Stefan; Van Grondelle, Rienk; Killian, J. Antoinette; Jones, Michael R.

    2014-01-01

    Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opp

  9. Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

    Science.gov (United States)

    Li, Yingying; Fu, Kehe; Gao, Shigang; Wu, Qiong; Fan, Lili; Li, Yaqian; Chen, Jie

    2013-01-01

    This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis) by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon-Wiener (H) and the Simpson (1-D) indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.

  10. Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

    Directory of Open Access Journals (Sweden)

    Yingying Li

    Full Text Available This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon-Wiener (H and the Simpson (1-D indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.

  11. Brassica RNA binding protein ERD4 is involved in conferring salt, drought tolerance and enhancing plant growth in Arabidopsis.

    Science.gov (United States)

    Rai, Archana N; Tamirisa, Srinath; Rao, K V; Kumar, Vinay; Suprasanna, P

    2016-03-01

    'Early responsive to dehydration' (ERD) genes are a group of plant genes having functional roles in plant stress tolerance and development. In this study, we have isolated and characterized a Brassica juncea 'ERD' gene (BjERD4) which encodes a novel RNA binding protein. The expression pattern of ERD4 analyzed under different stress conditions showed that transcript levels were increased with dehydration, sodium chloride, low temperature, heat, abscisic acid and salicylic acid treatments. The BjERD4 was found to be localized in the chloroplasts as revealed by Confocal microscopy studies. To study the function, transgenic Arabidopsis plants were generated and analyzed for various morphological and physiological parameters. The overexpressing transgenic lines showed significant increase in number of leaves with more leaf area and larger siliques as compared to wild type plants, whereas RNAi:ERD4 transgenic lines showed reduced leaf number, leaf area, dwarf phenotype and delayed seed germination. Transgenic Arabidopsis plants overexpressing BjERD4 gene also exhibited enhanced tolerance to dehydration and salt stresses, while the knockdown lines were susceptible as compared to wild type plants under similar stress conditions. It was observed that BjERD4 protein could bind RNA as evidenced by the gel-shift assay. The overall results of transcript analysis, RNA gel-shift assay, and transgenic expression, for the first time, show that the BjERD4 is involved in abiotic stress tolerance besides offering new clues about the possible roles of BjERD4 in plant growth and development.

  12. Brassica RNA binding protein ERD4 is involved in conferring salt, drought tolerance and enhancing plant growth in Arabidopsis.

    Science.gov (United States)

    Rai, Archana N; Tamirisa, Srinath; Rao, K V; Kumar, Vinay; Suprasanna, P

    2016-03-01

    'Early responsive to dehydration' (ERD) genes are a group of plant genes having functional roles in plant stress tolerance and development. In this study, we have isolated and characterized a Brassica juncea 'ERD' gene (BjERD4) which encodes a novel RNA binding protein. The expression pattern of ERD4 analyzed under different stress conditions showed that transcript levels were increased with dehydration, sodium chloride, low temperature, heat, abscisic acid and salicylic acid treatments. The BjERD4 was found to be localized in the chloroplasts as revealed by Confocal microscopy studies. To study the function, transgenic Arabidopsis plants were generated and analyzed for various morphological and physiological parameters. The overexpressing transgenic lines showed significant increase in number of leaves with more leaf area and larger siliques as compared to wild type plants, whereas RNAi:ERD4 transgenic lines showed reduced leaf number, leaf area, dwarf phenotype and delayed seed germination. Transgenic Arabidopsis plants overexpressing BjERD4 gene also exhibited enhanced tolerance to dehydration and salt stresses, while the knockdown lines were susceptible as compared to wild type plants under similar stress conditions. It was observed that BjERD4 protein could bind RNA as evidenced by the gel-shift assay. The overall results of transcript analysis, RNA gel-shift assay, and transgenic expression, for the first time, show that the BjERD4 is involved in abiotic stress tolerance besides offering new clues about the possible roles of BjERD4 in plant growth and development. PMID:26711633

  13. Multiple Metal Binding Domains Enhance the Zn(II) Selectivity of the Divalent Metal Ion Transporter AztA

    Energy Technology Data Exchange (ETDEWEB)

    Liu, T.; Reyes-Caballero, H.; Li, C.; Scott, R.A.; Giedroc, D.P.

    2009-06-03

    Transition metal-transporting P{sub 1B}-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We show that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA are functionally nonequivalent, but only with respect to zinc resistance. Inactivation of the a-MBD largely abrogates resistance to high intracellular Zn(II) levels, whereas inactivation of the b-MBD is not as deleterious. In contrast, inactivation of either the a- or b-MBD has little measurable impact on Cd(II) and Pb(II) resistance. The membrane proximal b-MBD binds Zn(II) with a higher affinity than the distal N-terminal a-MBD. Facile Zn(II)-specific intermolecular transfer from the a-MBD to the higher-affinity b-MBD is readily observed by {sup 1}H-{sup 15}N HSQC spectroscopy. Unlike Zn(II), Cd(II) and Pb(II) form saturated 1:1 S{sub 4} or S{sub 3}(O/N) complexes with AztA{sup aHbH}, where a single metal ion bridges the two MBDs. We propose that the tandem MBDs enhance Zn(II)-specific transport, while stabilizing a non-native inter-MBD Cd/Pb cross-linked structure that is a poor substrate and/or regulator for the transporter.

  14. CCAAT/enhancer binding protein {beta} deletion increases mitochondrial function and protects mice from LXR-induced hepatic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Shaikh M., E-mail: rmizanoor@hotmail.com [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Choudhury, Mahua; Janssen, Rachel C.; Baquero, Karalee C. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Miyazaki, Makoto [Division of Renal Diseases and Hypertension, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Friedman, Jacob E. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer LXR agonist activation increases liver TG accumulation by increasing lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta}{sup -/-} mouse prevents LXR activation-mediated induction of hepatic lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta} deletion increases mitochondrial transport chain function. Black-Right-Pointing-Pointer Beneficial effects of LXR activation on liver cholesterol metabolism did not change. Black-Right-Pointing-Pointer C/EBP{beta} inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBP{beta}) is an important regulator of liver gene expression but little is known about its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBP{beta} expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBP{beta} deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBP{beta}{sup -/-} mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBP{beta}{sup -/-} mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBP{beta} in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBP{beta} might therefore be an important therapeutic strategy to prevent LXR

  15. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

    Directory of Open Access Journals (Sweden)

    Barna Dey

    2009-05-01

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  16. Enhancing the Apoptotic Potential of Insulin-Like Growth Factor-Binding Protein-3 in Prostate Cancer by Modulation of CK2 Phosphorylation

    OpenAIRE

    Cobb, Laura J.; Mehta, Hemal; Cohen, Pinchas

    2009-01-01

    IGF-binding protein 3 (IGFBP-3) promotes apoptosis by both IGF-dependent and -independent mechanisms. We have previously reported that phosphorylation of IGFBP-3 (S156) by DNA-dependent protein kinase enhances its nuclear accumulation and is essential for its ability to interact with retinoid X receptor-α and induce apoptosis in cultured prostate cancer cells. Using specific chemical inhibitors and small interfering RNA, we demonstrate that preventing casein kinase 2 (CK2) activation enhanced...

  17. The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin

    Science.gov (United States)

    Gao, Feng; Gulay, Suna P.; Kasprzak, Wojciech; Dinman, Jonathan D.

    2013-01-01

    The Pea Enation Mosaic Virus (PEMV) 3′ translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5′-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5′ and 3′ PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3′ translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5′ hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5′ end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation. PMID:23986599

  18. Differential stimulation by CCAAT/enhancer-binding protein alpha isoforms of the estrogen-activated promoter of the very-low-density apolipoprotein II gene

    NARCIS (Netherlands)

    Calkhoven, CF; Snippe, L; Ab, G

    1997-01-01

    The transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBP alpha and C/EBP beta) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes, C/EBP alpha appears to be a critical regulator of genes involved in metabolic p

  19. Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss)CCAAT/enhancer binding protein genes and their responses to induction by GH in vitro and in vivo

    Science.gov (United States)

    CCAAT/enhancer-binding proteins (C/EBPs) are transcription factors consisting of six isoforms and play diverse physiological roles in vertebrates. In rainbow trout (Oncorhynchus mykiss), in addition to the reported C/EBPbeta1,we have isolated cDNA of four other isoforms, C/EBPalpha, C/EBPbeta2, C/E...

  20. Ectopic Expression of Hrf1 Enhances Bacterial Resistance via Regulation of Diterpene Phytoalexins, Silicon and Reactive Oxygen Species Burst in Rice

    Science.gov (United States)

    Zhong, Weigong; Yang, Jie; Okada, Kazunori; Yamane, Hisakazu; Zhang, Lei; Wang, Guang; Wang, Dong; Xiao, Shanshan; Chang, Shanshan; Qian, Guoliang; Liu, Fengquan

    2012-01-01

    Harpin proteins as elicitor derived from plant gram negative bacteria such as Xanthomonas oryzae pv. oryzae (Xoo), Erwinia amylovora induce disease resistance in plants by activating multiple defense responses. However, it is unclear whether phytoalexin production and ROS burst are involved in the disease resistance conferred by the expression of the harpinXoo protein in rice. In this article, ectopic expression of hrf1 in rice enhanced resistance to bacterial blight. Accompanying with the activation of genes related to the phytoalexin biosynthesis pathway in hrf1-transformed rice, phytoalexins quickly and consistently accumulated concurrent with the limitation of bacterial growth rate. Moreover, the hrf1-transformed rice showed an increased ability for ROS scavenging and decreased hydrogen peroxide (H2O2) concentration. Furthermore, the localization and relative quantification of silicon deposition in rice leaves was detected by scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometer (EDS). Finally, the transcript levels of defense response genes increased in transformed rice. These results show a correlation between Xoo resistance and phytoalexin production, H2O2, silicon deposition and defense gene expression in hrf1-transformed rice. These data are significant because they provide evidence for a better understanding the role of defense responses in the incompatible interaction between bacterial disease and hrf1-transformed plants. These data also supply an opportunity for generating nonspecific resistance to pathogens. PMID:22970151

  1. Ectopic expression of Hrf1 enhances bacterial resistance via regulation of diterpene phytoalexins, silicon and reactive oxygen species burst in rice.

    Directory of Open Access Journals (Sweden)

    Wenqi Li

    Full Text Available Harpin proteins as elicitor derived from plant gram negative bacteria such as Xanthomonas oryzae pv. oryzae (Xoo, Erwinia amylovora induce disease resistance in plants by activating multiple defense responses. However, it is unclear whether phytoalexin production and ROS burst are involved in the disease resistance conferred by the expression of the harpin(Xoo protein in rice. In this article, ectopic expression of hrf1 in rice enhanced resistance to bacterial blight. Accompanying with the activation of genes related to the phytoalexin biosynthesis pathway in hrf1-transformed rice, phytoalexins quickly and consistently accumulated concurrent with the limitation of bacterial growth rate. Moreover, the hrf1-transformed rice showed an increased ability for ROS scavenging and decreased hydrogen peroxide (H(2O(2 concentration. Furthermore, the localization and relative quantification of silicon deposition in rice leaves was detected by scanning electron microscopy (SEM and energy-dispersive X-ray spectrometer (EDS. Finally, the transcript levels of defense response genes increased in transformed rice. These results show a correlation between Xoo resistance and phytoalexin production, H(2O(2, silicon deposition and defense gene expression in hrf1-transformed rice. These data are significant because they provide evidence for a better understanding the role of defense responses in the incompatible interaction between bacterial disease and hrf1-transformed plants. These data also supply an opportunity for generating nonspecific resistance to pathogens.

  2. JC virus promoter/enhancers contain TATA box-associated Spi-B-binding sites that support early viral gene expression in primary astrocytes.

    Science.gov (United States)

    Marshall, Leslie J; Moore, Lisa D; Mirsky, Matthew M; Major, Eugene O

    2012-03-01

    JC virus (JCV) is the aetiological agent of the demyelinating disease progressive multifocal leukoencephalopathy, an AIDS defining illness and serious complication of mAb therapies. Initial infection probably occurs in childhood. In the working model of dissemination, virus persists in the kidney and lymphoid tissues until immune suppression/modulation causes reactivation and trafficking to the brain where JCV replicates in oligodendrocytes. JCV infection is regulated through binding of host factors such as Spi-B to, and sequence variation in the non-coding control region (NCCR). Although NCCR sequences differ between sites of persistence and pathogenesis, evidence suggests that the virus that initiates infection in the brain disseminates via B-cells derived from latently infected haematopoietic precursors in the bone marrow. Spi-B binds adjacent to TATA boxes in the promoter/enhancer of the PML-associated JCV Mad-1 and Mad-4 viruses but not the non-pathogenic, kidney-associated archetype. The Spi-B-binding site of Mad-1/Mad-4 differs from that of archetype by a single nucleotide, AAAAGGGAAGGGA to AAAAGGGAAGGTA. Point mutation of the Mad-1 Spi-B site reduced early viral protein large T-antigen expression by up to fourfold. Strikingly, the reverse mutation in the archetype NCCR increased large T-antigen expression by 10-fold. Interestingly, Spi-B protein binds the NCCR sequence flanking the viral promoter/enhancer, but these sites are not essential for early viral gene expression. The effect of mutating Spi-B-binding sites within the JCV promoter/enhancer on early viral gene expression strongly suggests a role for Spi-B binding to the viral promoter/enhancer in the activation of early viral gene expression.

  3. Computational identification of developmental enhancers:conservation and function of transcription factor binding-site clustersin drosophila melanogaster and drosophila psedoobscura

    Energy Technology Data Exchange (ETDEWEB)

    Berman, Benjamin P.; Pfeiffer, Barret D.; Laverty, Todd R.; Salzberg, Steven L.; Rubin, Gerald M.; Eisen, Michael B.; Celniker, SusanE.

    2004-08-06

    The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Measuring conservation of sequence features closely linked to function--such as binding-site clustering--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.

  4. First Analysis of a Bacterial Collagen-Binding Protein with Collagen Toolkits: Promiscuous Binding of YadA to Collagens May Explain How YadA Interferes with Host Processes▿ †

    OpenAIRE

    Jack C. Leo; Elovaara, Heli; Bihan, Dominique; Pugh, Nicholas; Kilpinen, Sami K.; Raynal, Nicolas; Skurnik, Mikael; Farndale, Richard W.; Goldman, Adrian

    2010-01-01

    The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. Yad...

  5. Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

    OpenAIRE

    Naz, Neveda; Mills, Dominic C.; Wren, Brendan W.; Dorrell, Nick

    2013-01-01

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusi...

  6. Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms†

    OpenAIRE

    Burmølle, Mette; Webb, Jeremy S; Rao, Dhana; Hansen, Lars H.; Sørensen, Søren J.; Kjelleberg, Staffan

    2006-01-01

    Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosph...

  7. The role of silicon in enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice.

    Science.gov (United States)

    Song, Alin; Xue, Gaofeng; Cui, Peiyuan; Fan, Fenliang; Liu, Hongfang; Yin, Chang; Sun, Wanchun; Liang, Yongchao

    2016-04-19

    Here we report for the first time that bacterial blight of rice can be alleviated by silicon (Si) added. In both inoculated and uninoculated plants, shoot dry weight was significantly higher in the +Si plants than in the -Si plants. A soil-cultured trial showed that disease severity was 24.3% lower in the Si-amended plants than in the non-Si-amended plants. Plants that were switched from -Si to +Si nutrient solution and simultaneously inoculated with Xoo also exhibited the same high resistance to bacterial blight as the plants that were treated continuously with Si, with control efficiencies of 52.8 and 62.9%, respectively. Moreover, total concentrations of soluble phenolics and lignin in rice leaves were significantly higher in the +Si plants than in the -Si plants. Polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities in rice leaves were observed to be higher in the +Si plants than in the -Si plants. The expression levels of Os03g0109600, Prla, Rcht2 and Lox2osPil, were also higher in +Si plants than in -Si plants post-inoculation during the experimental time. Addition of Si resulted in increased Pal transcription, and inhibited CatA and Os03g0126000 expression in the earlier and later stages of bacterial inoculation, respectively.

  8. The role of silicon in enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice.

    Science.gov (United States)

    Song, Alin; Xue, Gaofeng; Cui, Peiyuan; Fan, Fenliang; Liu, Hongfang; Yin, Chang; Sun, Wanchun; Liang, Yongchao

    2016-01-01

    Here we report for the first time that bacterial blight of rice can be alleviated by silicon (Si) added. In both inoculated and uninoculated plants, shoot dry weight was significantly higher in the +Si plants than in the -Si plants. A soil-cultured trial showed that disease severity was 24.3% lower in the Si-amended plants than in the non-Si-amended plants. Plants that were switched from -Si to +Si nutrient solution and simultaneously inoculated with Xoo also exhibited the same high resistance to bacterial blight as the plants that were treated continuously with Si, with control efficiencies of 52.8 and 62.9%, respectively. Moreover, total concentrations of soluble phenolics and lignin in rice leaves were significantly higher in the +Si plants than in the -Si plants. Polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities in rice leaves were observed to be higher in the +Si plants than in the -Si plants. The expression levels of Os03g0109600, Prla, Rcht2 and Lox2osPil, were also higher in +Si plants than in -Si plants post-inoculation during the experimental time. Addition of Si resulted in increased Pal transcription, and inhibited CatA and Os03g0126000 expression in the earlier and later stages of bacterial inoculation, respectively. PMID:27091552

  9. The role of silicon in enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice

    Science.gov (United States)

    Song, Alin; Xue, Gaofeng; Cui, Peiyuan; Fan, Fenliang; Liu, Hongfang; Yin, Chang; Sun, Wanchun; Liang, Yongchao

    2016-01-01

    Here we report for the first time that bacterial blight of rice can be alleviated by silicon (Si) added. In both inoculated and uninoculated plants, shoot dry weight was significantly higher in the +Si plants than in the −Si plants. A soil-cultured trial showed that disease severity was 24.3% lower in the Si-amended plants than in the non-Si-amended plants. Plants that were switched from −Si to +Si nutrient solution and simultaneously inoculated with Xoo also exhibited the same high resistance to bacterial blight as the plants that were treated continuously with Si, with control efficiencies of 52.8 and 62.9%, respectively. Moreover, total concentrations of soluble phenolics and lignin in rice leaves were significantly higher in the +Si plants than in the −Si plants. Polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities in rice leaves were observed to be higher in the +Si plants than in the −Si plants. The expression levels of Os03g0109600, Prla, Rcht2 and Lox2osPil, were also higher in +Si plants than in −Si plants post-inoculation during the experimental time. Addition of Si resulted in increased Pal transcription, and inhibited CatA and Os03g0126000 expression in the earlier and later stages of bacterial inoculation, respectively. PMID:27091552

  10. Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a.

    Science.gov (United States)

    Chu, Yu-Yi; Ko, Chiung-Yuan; Wang, Wei-Jan; Wang, Shao-Ming; Gean, Po-Wu; Kuo, Yu-Min; Wang, Ju-Ming

    2016-08-01

    The progression of Alzheimer's disease (AD) has been associated with astrocytes-induced neuroinflammation. However, the detailed mechanism of astrocytes associated with learning impairments and neuronal loss in AD is poorly defined. Here, we provide novel evidences that astrocytic miR-135a is critical for neuronal viability and spatial learning ability in vivo. The AppTg/Cebpd (-/-) mice showed a spatial learning improvement compared with the APPswe/PS1/E9 bigenic (AppTg) mice. miR-135a was found to be a CCAAT/enhancer binding protein δ (CEBPD) responsive miRNA and can repress the transcription of thrombospondin 1 (THBS1) / Thbs1 (mouse) via its 3'-untranslated region (3'UTR). We used different experimental approaches to attenuate the expression of CEBPD/Cebpd (mouse) or miR-135a in astrocytes and found the following results: increase in THBS1/Thbs1 expression, decrease in neuronal apoptosis, and increase in growth of neurites. Importantly, injection of miR-135a antagonist (AM135a) into the brain of AppTg mice was found to prevent neuronal apoptosis and improved the spatial learning ability. Together, our findings demonstrate a critical function for the astrocytic CEBPD, and point to miR-135a antagonist as an attractive therapeutic target for the treatment of Alzheimer's disease. PMID:26208701

  11. Human α-defensin expression is not dependent on CCAAT/enhancer binding protein-ε in a murine model.

    Directory of Open Access Journals (Sweden)

    Andreas Glenthøj

    Full Text Available Specific granule deficiency (SGD is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-ε (C/EBP-ε gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, α-defensins, also known as human neutrophil peptides (HNPs. In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-ε. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-ε in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe-/- mouse to study the in vivo significance of C/EBP-ε for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-ε in these mice. Transduction of C/EBP-ε into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions.

  12. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    International Nuclear Information System (INIS)

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning

  13. Acute heat stress and thermal acclimation induce CCAAT/enhancer-binding protein delta in the goby Gillichthys mirabilis.

    Science.gov (United States)

    Buckley, Bradley A

    2011-08-01

    Members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors have regulatory control over numerous processes related to cell fate determination, including differentiation, proliferation, cell cycle arrest and apoptosis. In mammals, abnormalities in the expression of some isoforms of C/EBPs are pathogenic and are implicated as being involved in myeloid leukemia and breast cancers. Next to nothing is known about their regulation, function or stress-responsiveness in poikilotherms. Here, both acute heat stress and thermal acclimation were demonstrated to induce the expression of one isoform, C/EBP-δ, in the liver, white muscle and gill of the eurythermal estuarine goby, Gillichthys mirabilis. The established role of C/EBP-δ in causing cell cycle arrest and/or promoting apoptosis in other vertebrates suggests that the heat-inducibility of this protein in poikilotherms may be part of the conserved cellular stress response with the hypothesized role of causing temporary cessation of cell growth and/or programmed cell death during bouts of environmental stress. The observed regulation of c/ebp-δ during hyperthermia represents a novel, heat-inducible signaling pathway in fishes. PMID:21442321

  14. Handheld chem/biosensor using extreme conformational changes in designed binding proteins to enhance surface plasmon resonance (SPR)

    Science.gov (United States)

    Lepak, Lori A.; Schnatz, Peter; Bendoym, Igor; Kosciolek, Derek; Koder, Ronald; Crouse, David T.

    2016-05-01

    We present research results centered on development of a highly sensitive handheld chem/biosensor device using a novel class of engineered proteins, designed to undergo extreme conformational changes upon binding their target, which in turn cause extreme changes in refractive index in the protein layer. These proteins are attached to a detector chip with a structured metasurface, to translate the refractive index change into an enhanced shift in surface plasmon resonances (SPR), thereby improving the sensitivity of the overall detector relatively to current commercially available SPR systems. Theoretical calculations have demonstrated the potential of the conformational changes in the engineered proteins to provide the desired change in refractive index. A plasmonic chip with a simple grating metasurface structure was designed to maximize the SPR shift. A prototype chip and a prototype for the overall device housing were fabricated with the inclusion of all other required (commercially available) optical components. The proposed device holds considerable promise as a low-cost, highly sensitive, field-deployable detection system for chemical and biological toxins.

  15. CCAAT/enhancer-binding protein CEBP-2 controls fat consumption and fatty acid desaturation in Caenorhabditis elegans.

    Science.gov (United States)

    Xu, Xiao-Ying; Hu, Jian-Ping; Wu, Meng-Meng; Wang, Li-Shun; Fang, Ning-Yuan

    Mammalian CCAAT/enhancer-binding proteins (C/EBPs) are generally known as regulators in adipocyte differentiation. However, more understanding of the role of C/EBPs in lipid and glucose metabolism remains to be discovered. In this study, we verified the effect of CEBP-2, the homolog of CEBPs, on fat storage in Caenorhabditis elegans. Expressions of 85 genes that encode the major enzymes in energy metabolic pathways were then screened in cebp-2-deficient worms using a quantitative real-time polymerase chain reaction (QRT-PCR). Our data implied that loss of function of CEBP-2 displayed a low-fat phenotype in C. elegans owing to increased expression of ech-1.1 and decreased expression of fat-5. Our findings indicated that cebp-2 controls total body fat content by governing fatty acid mitochondrial β-oxidation and desaturation in C. elegans. These data provide insights into how C/EBPs may affect lipid metabolism in mammals in addition to regulating adipocyte differentiation.

  16. CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells.

    Science.gov (United States)

    Santangelo, Carmela; Varì, Rosaria; Scazzocchio, Beatrice; Filesi, Carmelina; D'Archivio, Massimo; Giovannini, Claudio; Masella, Roberta

    2011-09-01

    Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the

  17. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    Science.gov (United States)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  18. Enhanced expression of lmb gene encoding laminin-binding protein in Streptococcus agalactiae strains harboring IS1548 in scpB-lmb intergenic region.

    Directory of Open Access Journals (Sweden)

    Rim Al Safadi

    Full Text Available Group B streptococcus (GBS is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs: the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs. IS1548 was mostly found (72.2% in CC19 serotype III strains recovered more specifically (92.3% from neonatal meningitis. GBSi1 was principally found (70.6% in CC17 strains, mostly (94.4% of serotype III, but also (15.7% in CC19 strains, mostly (87.5% of serotype II. No MGE was found in most strains of the other CCs (76.0%, notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001. Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P< or =0.001. Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P<0.001. By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold, the binding ability to laminin (6.2-fold, and the expression of Lmb protein (5.0-fold. These data highlight the importance of MGEs in bacterial virulence and demonstrate the up-regulation of lmb gene by IS

  19. Building Enhancers From the Ground Up: A Synthetic Biology Approach

    OpenAIRE

    Amit, Roee; Garcia, Hernan G.; Phillips, Rob; Fraser, Scott E.

    2011-01-01

    A challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhancers, combining a driver, transcription factor binding sites, and a poised polymerase to create synthetic modular enhancers. Our findings suggest that enhancer-based transcriptional control depends critically and quantitatively on DNA looping, leading to complex regulatory effects when the enhancer ca...

  20. Crystal Structures of the Staphylococcal Toxin SSL5 in Complex With Sialyl-Lewis X Reveal a Conserved Binding Site That Shares Common Features With Viral And Bacterial Sialic Acid-Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Baker, H.M.; Basu, I.; Chung, M.C.; Caradoc-Davies, T.; Fraser, J.D.; Baker, E.N.

    2009-06-02

    Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.

  1. Enhancement of Urinary Bladder Carcinogenesis by the Role of Chronic Bacterial Infection-induced Inflammation (Imunnohistochemical and Biochemical studies

    Directory of Open Access Journals (Sweden)

    Gabri MS*, Ashmawy AM**, Ibrahim MA*, Hosny RM

    2012-07-01

    Full Text Available Background: Bacterial infections traditionally have not been considered major causes of cancer. Recently, however, bacteria have been linked to cancer by two mechanisms: induction of chronic inflammation and production of carcinogenic bacterial metabolites. The most specific example of the inflammatory mechanism of carcinogenesis is Escherichia coli infection. E. coli has been epidemiologically linked to urothelial carcinoma of the urinary bladder by its propensity to cause lifelong inflammation. This inflammation is in turn thought to cause cancer by inducing cell proliferation and production of mutagenic free radicals and N-nitroso compounds.Material and methods: After each 3, 6 and 9 months of daily oral administration of dibutyl amine (DBA plus sodium nitrate (nitrosamine precursors in drinking water, curcuma in grinding diet and bladder injection with E. coli, rats were sacrificed. The excited bladder were dissected, processed and stained with H&E and anti-Ki67 immunohistochemical stains. This was followed by Elisa for caspse-3 and statistical analysis.Results: The current results indicated that E. coli infection in the bladder tissues increases the carcinogenic ability of nitrosamine precursors through caused marked alteration in the form hyperplastic, dysplastic and metaplastic urothelium. Also, there was a statistically significant increase in ki67 immunoreactivity in urothelium. However, a statistically significant decrease in the concentration of caspase-3 in bladder tissue consequently caused the process of carcinogenesis. All these changes were less marked after curcuma treatment when compared with the group that not treated with curcuma. Conclusion: Bacterial infection of the urinary bladder may play a major additive and possible role in bladder carcinogenesis. Rhizome of curcuma may have a protective action during induction of urinary bladder tumors.

  2. Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

    Science.gov (United States)

    Coleman, Timothy P; Tran, Quincy; Roesser, James R

    2003-01-27

    The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

  3. Liposomal co-entrapment of CD40mAb induces enhanced IgG responses against bacterial polysaccharide and protein.

    Directory of Open Access Journals (Sweden)

    Caterina Hatzifoti

    Full Text Available BACKGROUND: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen. Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required. We explore here an alternative to chemical conjugation, involving the co-encapsulation of CD40 antibody and antigens in liposomal vehicles. METHODOLOGY/PRINCIPAL FINDINGS: Anti-mouse CD40 mAb or isotype control mAb were co-entrapped individually in cationic liposomal vehicles with pneumococcal polysaccharides or diphtheria and tetanus toxoids. Retention of CD40 binding activity upon liposomal entrapment was assessed by ELISA and flow cytometry. After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA. Simple co-encapsulation of CD40 antibody allowed for the retention of CD40 binding on the liposome surface, and also produced vaccines with enhanced imunogenicity. Antibody responses against both co-entrapped protein in the form of tetanus toxoid, and Streptococcus pneumoniae capsular polysaccharide, were enhanced by co-encapsulation with CD40 antibody. Surprisingly, liposomal encapsulation also appeared to decrease the toxicity of high doses of CD40 antibody as assessed by the degree of splenomegaly induced. CONCLUSIONS/SIGNIFICANCE: Liposomal co-encapsulation with CD40 antibody may represent a practical means of producing more immunogenic multivalent vaccines and inducing IgG responses against polysaccharides without the need for conjugation.

  4. Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

    Science.gov (United States)

    Heuveling, Johanna; Frochaux, Violette; Ziomkowska, Joanna; Wawrzinek, Robert; Wessig, Pablo; Herrmann, Andreas; Schneider, Erwin

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

  5. Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding

    Science.gov (United States)

    Lee, Jin Hee; Park, Jin Won; Moon, Seung Hwan; Cho, Young Seok; Choe, Yearn Seong; Lee, Kyung-Han

    2016-01-01

    To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH. PMID:27479079

  6. Vimentin in Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs. IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  7. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  8. Bridged bis(beta-cyclodextrin)s possessing coordinated metal center(s) and their inclusion complexation behavior with model substrates: enhanced molecular binding ability by multiple recognition.

    Science.gov (United States)

    Liu, Y; Chen, Y; Li, L; Zhang, H Y; Liu, S X; Guan, X D

    2001-12-14

    To investigate quantitatively the cooperative binding ability of several beta-cyclodextrin oligomers bearing single or multiligated metal center(s), the inclusion complexation behavior of four bis(beta-cyclodextrin)s (2-5) linked by 2,2'-bipyridine-4,4'-dicarboxy tethers and their copper(II) complexes (6-9) with representative dye guests, i.e., methyl orange (MO), acridine red (AR), rhodamine B (RhB), ammonium 8-anilino-1-naphthalenesulfonic acid (ANS), and sodium 6-(p-toludino)-2-naphthalenesulfonate (TNS), have been examined in aqueous solution at 25 degrees C by means of UV-vis, circular dichroism, fluorescence, and 2D NMR spectroscopy. The results obtained indicate that bis(beta-cyclodextrin)s 2-5 can associate with one or three copper(II) ion(s) producing 2:1 or 2:3 bis(beta-cyclodextrin)-copper(II) complexes. These metal-ligated oligo(beta-cyclodextrin)s can bind two model substrates to form intramolecular 2:2 host-guest inclusion complexes and thus significantly enhance the original binding abilities of parent beta-cyclodextrin and bis(beta-cyclodextrin) toward model substrates through the cooperative binding of two guest molecules by four tethered cyclodextrin moieties, as well as the additional binding effect supplied by ligated metal center(s). Host 6 showed the highest enhancement of the stability constant, up to 38.3 times for ANS as compared with parent beta-cyclodextrin. The molecular binding mode and stability constant of substrates by bridged bis- and oligo(beta-cyclodextrin)s 2-9 are discussed from the viewpoint of the size/shape-fit interaction and molecular multiple recognition between host and guest.

  9. Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.

    Science.gov (United States)

    Naz, Neveda; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

    2013-01-01

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells. PMID:24192850

  10. Enhanced striatial /sup 3/H-spiroperidol binding induced by chronic haloperidol treatment inhibited by peptides administered during the withdrawal phase

    Energy Technology Data Exchange (ETDEWEB)

    Bhargava, H.N.

    1984-02-27

    Chronic intragastric administration of haloperidol (1.5 mg/kg/day) for 21 days followed by a 3-day withdrawal period resulted in the development of enhanced locomotor activity response to apomorphine, and an increase in the number of binding sites for /sup 3/H-spiroperidol in the striatal membranes of the rat brain. Subcutaneous administration of Pro-Leu-Gly-NH/sub 2/ or cyclo-(Leu-Gly) in doses of 2 mg/kg/day given for 3-days after termination of haloperidol treatment inhibited the enhanced response to apomorphine, as well as the increases in the number of /sup 3/H-spiroperidol binding sites in the striatum. If indeed, the supersensitivity of striatal dopamine receptors is one of the mechanisms in the development of tardive dyskinesia symptoms, the present results suggest that the above peptides may be helpful in ameliorating some of the symptoms of tardive dyskinesia induced by neuroleptic drugs. 31 references, 3 figures.

  11. Pili play an important role in enhancing the bacterial clearance from the middle ear in a mouse model of acute otitis media with Moraxella catarrhalis.

    Science.gov (United States)

    Kawano, Toshiaki; Hirano, Takashi; Kodama, Satoru; Mitsui, Marcelo Takahiro; Ahmed, Kamruddin; Nishizono, Akira; Suzuki, Masashi

    2013-03-01

    Moraxella catarrhalis is a Gram-negative aerobic diplococcus that is currently the third most frequent cause of bacterial acute otitis media (AOM) in children. In this study, we developed an experimental murine AOM model by inoculating M. catarrhalis in the middle ear bulla and studied the local response to this inoculation, and modulation of its course by the pili of M. catarrhalis. The pili-positive and pili-negative M. catarrhalis showed differences in bacterial clearance and infiltration of inflammatory cells in the middle ear. Pili-negative M. catarrhalis induced a more delayed and prolonged immune response in the middle ear than that of pili-positive M. catarrhalis. TLR2, -4, -5 and -9 mRNA expression was upregulated in neutrophils that infiltrated the middle ear cavity during AOM caused by both pili-positive and pili-negative bacteria. TLR5 mRNA expression and TLR5 protein in the neutrophils were induced more robustly by pili-positive M. catarrhalis. This immune response is likely to be related to neutrophil function such as toll-like 5-dependent phagocytosis. Our results show that mice may provide a useful AOM model for studying the role of M. catarrhalis. Furthermore, we show that pili play an important role in enhancing M. catarrhalis clearance from the middle ear that is probably mediated through neutrophil-dependent TLR5 signaling.

  12. Toll-Like Receptor 9 Enhances Bacterial Clearance and Limits Lung Consolidation in Murine Pneumonia Caused by Methicillin-Resistant Staphylococcus aureus

    Science.gov (United States)

    van der Meer, Anne Jan; Achouiti, Achmed; van der Ende, Arie; Soussan, Aicha Ait; Florquin, Sandrine; de Vos, Alex; Zeerleder, Sacha S; van der Poll, Tom

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in pneumonia associated with severe lung damage. Tissue injury causes release of damage-associated molecular patterns (DAMPs), which may perpetuate inflammation. DNA has been implicated as a DAMP that activates inflammation through Toll-like receptor 9 (TLR9). The aim of this study was to evaluate the role of TLR9 in MRSA pneumonia. Wild-type (Wt) and TLR9 knockout (tlr9−/−) mice were infected intranasally with MRSA USA300 (BK 11540) (5E7 CFU) and euthanized at 6, 24, 48 or 72 h for analyses. MRSA pneumonia was associated with profound release of cell-free host DNA in the airways, as reflected by increases in nucleosome and DNA levels in bronchoalveolar lavage fluid (BALF), accompanied by transient detection of pathogen DNA in MRSA-free BALF supernatants. In BALF, as compared with Wt mice, tlr9−/− mice showed reduced tumor necrosis factor α and IL-6 levels at 6 h and reduced bacterial clearance at 6 and 24 h postinfection. Furthermore, tlr9−/− mice exhibited a greater influx of neutrophils in BALF and increased lung consolidation at 24 and 48 h. This study demonstrates the release of host- and pathogen-derived TLR9 ligands (DNA) into the alveolar space after infection with MRSA via the airways and suggests that TLR9 has proinflammatory effects during MRSA pneumonia associated with enhanced bacterial clearance and limitation of lung consolidation. PMID:27508882

  13. OppA of Listeria monocytogenes, an Oligopeptide-Binding Protein Required for Bacterial Growth at Low Temperature and Involved in Intracellular Survival

    OpenAIRE

    Borezee, Elise; Pellegrini, Elisabeth; Berche, Patrick

    2000-01-01

    We identified a new oligopeptide permease operon in the pathogen Listeria monocytogenes. This opp operon consists of five genes (oppA, oppB, oppC, oppD, and oppF) and displays the same genetic organization as those of several bacterial species. The first gene of this operon, oppA, encodes a 62-kDa protein sharing 33% identity with OppA of Bacillus subtilis and is expressed predominantly during exponential growth. The function of oppA was studied by constructing an oppA deletion mutant. The ph...

  14. Cooperative Model of Bacterial Sensing

    CERN Document Server

    Shi, Y; Shi, Yu; Duke, Thomas

    1998-01-01

    Bacterial chemotaxis is controlled by the signalling of a cluster of receptors. A cooperative model is presented, in which coupling between neighbouring receptor dimers enhances the sensitivity with which stimuli can be detected, without diminishing the range of chemoeffector concentration over which chemotaxis can operate. Individual receptor dimers have two stable conformational states: one active, one inactive. Noise gives rise to a distribution between these states, with the probability influenced by ligand binding, and also by the conformational states of adjacent receptor dimers. The two-state model is solved, based on an equivalence with the Ising model in a randomly distributed magnetic field. The model has only two effective parameters, and unifies a number of experimental findings. According to the value of the parameter comparing coupling and noise, the signal can be arbitrarily sensitive to changes in the fraction of receptor dimers to which ligand is bound. The counteracting effect of a change of...

  15. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Theerthankar Das

    Full Text Available Calcium (Ca(2+ has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG values in iTC data confirmed that the interaction between DNA and Ca(2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.

  16. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    Science.gov (United States)

    Das, Theerthankar; Sehar, Shama; Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

  17. CCAAT/enhancer binding proteins alpha and epsilon cooperate with all-trans retinoic acid in therapy but differ in their antileukemic activities

    OpenAIRE

    Lee, Young-jin; Jones, Letetia C.; Timchenko, Nikolai A.; Perrotti, Danilo; Tenen, Daniel G; Kogan, Scott C.

    2006-01-01

    CCAAT/enhancer binding proteins (C/EBPs) play critical roles in myelopoiesis. Dysregulation of these proteins likely contributes to the pathogenesis of myeloid disorders characterized by a block in granulopoiesis. In one such disease, acute promyelocytic leukemia (APL), a promyelocytic leukemia–retinoic acid receptor α (PML-RARα) fusion protein is expressed as a result of a t(15;17) chromosomal translocation. Treatment of PML-RARα leukemic cells with all-trans retinoic acid (ATRA) causes them...

  18. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  19. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  20. Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene.

    Science.gov (United States)

    Maxson, J M; Woodson, W R

    1996-07-01

    Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.

  1. Identification of a Distinct Substrate-binding Domain in the Bacterial Cysteine Methyltransferase Effectors NleE and OspZ.

    Science.gov (United States)

    Zhang, Ying; Mühlen, Sabrina; Oates, Clare V; Pearson, Jaclyn S; Hartland, Elizabeth L

    2016-09-16

    The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif (49)GITR(52), was critical for TAB2 and TAB3 binding. NleE mutants lacking (49)GITR(52) were unable to methylate TAB3, and wild type NleE but not NleE(49AAAA52) where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the (49)GITR(52) motif. Ectopic expression of an N-terminal fragment of NleE (NleE(34-52)) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the (49)GITR(52) motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response. PMID:27445336

  2. Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography

    OpenAIRE

    Ravaud, Stephanie; Gouet, Patrice; Haser, Richard; Aghajari, Nushin

    2003-01-01

    The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted ...

  3. Defective bacterial clearance is responsible for the enhanced lung pathology characteristic of Mannheimia haemolytica pneumonia in bighorn sheep

    Science.gov (United States)

    The molecular and cellular basis for the enhanced lung pathology and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS, Ovis canadenesis), in comparison to domestic sheep (DS, Ovis aries), is not clear. Polymorphonuclear leukocytes (PMNs) of BHS are four- to eight-fold more susceptibl...

  4. Allosteric enhancers, allosteric agonists and ago-allosteric modulators: where do they bind and how do they act?

    DEFF Research Database (Denmark)

    Schwartz, Thue W; Holst, Birgitte

    2007-01-01

    Many small-molecule agonists also display allosteric properties. Such ago-allosteric modulators act as co-agonists, providing additive efficacy--instead of partial antagonism--and they can affect--and often improve--the potency of the endogenous agonist. Surprisingly, the apparent binding sites...... different binding modes. In another, dimeric, receptor scenario, the endogenous agonist binds to one protomer while the ago-allosteric modulator binds to the other, 'allosteric' protomer. It is suggested that testing for ago-allosteric properties should be an integral part of the agonist drug discovery...... process because a compound that acts with--rather than against--the endogenous agonist could be an optimal agonist drug....

  5. Kinetics of H+ ion binding by the P+QA-state of bacterial photosynthetic reaction centers: rate limitation within the protein.

    Science.gov (United States)

    Maróti, P; Wraight, C A

    1997-01-01

    The kinetics of flash-induced H+ ion binding by isolated reaction centers (RCs) of Rhodobacter sphaeroides, strain R-26, were measured, using pH indicators and conductimetry, in the presence of terbutryn to block electron transfer between the primary and secondary quinones (QA and QB), and in the absence of exogenous electron donors to the oxidized primary donor, P+, i.e., the P+QA-state. Under these conditions, proton binding by RCs is to the protein rather than to any of the cofactors. After light activation to form P+QA-, the kinetics of proton binding were monoexponential at all pH values studied. At neutral pH, the apparent bimolecular rate constant was close to the diffusional limit for proton transfer in aqueous solution (approximately 10(11) M-1 s-1), but increased significantly in the alkaline pH range (e.g., 2 x 10(13) M-1 s-1 at pH 10). The average slope of the pH dependence was -0.4 instead of -1.0, as might be expected for a H+ diffusion-controlled process. High activation energy (0.54 eV at pH 8.0) and weak viscosity dependence showed that H+ ion uptake by RCs is not limited by diffusion. The salt dependence of the H+ ion binding rate and the pK values of the protonatable amino acid residues of the reaction center implicated surface charge influences, and Gouy-Chapman theory provided a workable description of the ionic effects as arising from modulation of the pH at the surface of the RC. Incubation in D2O caused small increases in the pKs of the protonatable groups and a small, pH (pD)-dependent slowing of the binding rate. The salt, pH, temperature, viscosity, and D2O dependences of the proton uptake by RCs in the P+QA- state were accounted for by three considerations: 1) parallel pathways of H+ delivery to the RC, contributing to the observed (net) H+ disappearance; 2) rate limitation of the protonation of target groups within the protein by conformational dynamics; and 3) electrostatic influences of charged groups in the protein, via the surface p

  6. Kinetics of H+ ion binding by the P+QA-state of bacterial photosynthetic reaction centers: rate limitation within the protein.

    Science.gov (United States)

    Maróti, P; Wraight, C A

    1997-07-01

    The kinetics of flash-induced H+ ion binding by isolated reaction centers (RCs) of Rhodobacter sphaeroides, strain R-26, were measured, using pH indicators and conductimetry, in the presence of terbutryn to block electron transfer between the primary and secondary quinones (QA and QB), and in the absence of exogenous electron donors to the oxidized primary donor, P+, i.e., the P+QA-state. Under these conditions, proton binding by RCs is to the protein rather than to any of the cofactors. After light activation to form P+QA-, the kinetics of proton binding were monoexponential at all pH values studied. At neutral pH, the apparent bimolecular rate constant was close to the diffusional limit for proton transfer in aqueous solution (approximately 10(11) M-1 s-1), but increased significantly in the alkaline pH range (e.g., 2 x 10(13) M-1 s-1 at pH 10). The average slope of the pH dependence was -0.4 instead of -1.0, as might be expected for a H+ diffusion-controlled process. High activation energy (0.54 eV at pH 8.0) and weak viscosity dependence showed that H+ ion uptake by RCs is not limited by diffusion. The salt dependence of the H+ ion binding rate and the pK values of the protonatable amino acid residues of the reaction center implicated surface charge influences, and Gouy-Chapman theory provided a workable description of the ionic effects as arising from modulation of the pH at the surface of the RC. Incubation in D2O caused small increases in the pKs of the protonatable groups and a small, pH (pD)-dependent slowing of the binding rate. The salt, pH, temperature, viscosity, and D2O dependences of the proton uptake by RCs in the P+QA- state were accounted for by three considerations: 1) parallel pathways of H+ delivery to the RC, contributing to the observed (net) H+ disappearance; 2) rate limitation of the protonation of target groups within the protein by conformational dynamics; and 3) electrostatic influences of charged groups in the protein, via the surface pH.

  7. A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

    OpenAIRE

    Fox, M E; Feldman, B. J.; Chu, G.

    1994-01-01

    DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2....

  8. Nanocatalysts promote Streptococcus mutans biofilm matrix degradation and enhance bacterial killing to suppress dental caries in vivo.

    Science.gov (United States)

    Gao, Lizeng; Liu, Yuan; Kim, Dongyeop; Li, Yong; Hwang, Geelsu; Naha, Pratap C; Cormode, David P; Koo, Hyun

    2016-09-01

    Dental biofilms (known as plaque) are notoriously difficult to remove or treat because the bacteria can be enmeshed in a protective extracellular matrix. It can also create highly acidic microenvironments that cause acid-dissolution of enamel-apatite on teeth, leading to the onset of dental caries. Current antimicrobial agents are incapable of disrupting the matrix and thereby fail to efficiently kill the microbes within plaque-biofilms. Here, we report a novel strategy to control plaque-biofilms using catalytic nanoparticles (CAT-NP) with peroxidase-like activity that trigger extracellular matrix degradation and cause bacterial death within acidic niches of caries-causing biofilm. CAT-NP containing biocompatible Fe3O4 were developed to catalyze H2O2 to generate free-radicals in situ that simultaneously degrade the biofilm matrix and rapidly kill the embedded bacteria with exceptional efficacy (>5-log reduction of cell-viability). Moreover, it displays an additional property of reducing apatite demineralization in acidic conditions. Using 1-min topical daily treatments akin to a clinical situation, we demonstrate that CAT-NP in combination with H2O2 effectively suppress the onset and severity of dental caries while sparing normal tissues in vivo. Our results reveal the potential to exploit nanocatalysts with enzyme-like activity as a potent alternative approach for treatment of a prevalent biofilm-associated oral disease. PMID:27294544

  9. Rhizospheric Bacterial Strain Brevibacterium casei MH8a Colonizes Plant Tissues and Enhances Cd, Zn, Cu Phytoextraction by White Mustard

    OpenAIRE

    Płociniczak, Tomasz; Sinkkonen, Aki; Romantschuk, Martin; Sułowicz, Sławomir; Piotrowska-Seget, Zofia

    2016-01-01

    Environmental pollution by heavy metals has become a serious problem in the world. Phytoextraction, which is one of the plant-based technologies, has attracted the most attention for the bioremediation of soils polluted with these contaminants. The aim of this study was to determine whether the multiple-tolerant bacterium, Brevibacterium casei MH8a isolated from the heavy metal-contaminated rhizosphere soil of Sinapis alba L., is able to promote plant growth and enhance Cd, Zn, and Cu uptake ...

  10. Dietary Fiber and Bacterial SCFA Enhance Oral Tolerance and Protect against Food Allergy through Diverse Cellular Pathways

    Directory of Open Access Journals (Sweden)

    Jian Tan

    2016-06-01

    Full Text Available The incidence of food allergies in western countries has increased dramatically in recent decades. Tolerance to food antigens relies on mucosal CD103+ dendritic cells (DCs, which promote differentiation of regulatory T (Treg cells. We show that high-fiber feeding in mice improved oral tolerance and protected from food allergy. High-fiber feeding reshaped gut microbial ecology and increased the release of short-chain fatty acids (SCFAs, particularly acetate and butyrate. High-fiber feeding enhanced oral tolerance and protected against food allergy by enhancing retinal dehydrogenase activity in CD103+ DC. This protection depended on vitamin A in the diet. This feeding regimen also boosted IgA production and enhanced T follicular helper and mucosal germinal center responses. Mice lacking GPR43 or GPR109A, receptors for SCFAs, showed exacerbated food allergy and fewer CD103+ DCs. Dietary elements, including fiber and vitamin A, therefore regulate numerous protective pathways in the gastrointestinal tract, necessary for immune non-responsiveness to food antigens.

  11. Dietary Fiber and Bacterial SCFA Enhance Oral Tolerance and Protect against Food Allergy through Diverse Cellular Pathways.

    Science.gov (United States)

    Tan, Jian; McKenzie, Craig; Vuillermin, Peter J; Goverse, Gera; Vinuesa, Carola G; Mebius, Reina E; Macia, Laurence; Mackay, Charles R

    2016-06-21

    The incidence of food allergies in western countries has increased dramatically in recent decades. Tolerance to food antigens relies on mucosal CD103(+) dendritic cells (DCs), which promote differentiation of regulatory T (Treg) cells. We show that high-fiber feeding in mice improved oral tolerance and protected from food allergy. High-fiber feeding reshaped gut microbial ecology and increased the release of short-chain fatty acids (SCFAs), particularly acetate and butyrate. High-fiber feeding enhanced oral tolerance and protected against food allergy by enhancing retinal dehydrogenase activity in CD103(+) DC. This protection depended on vitamin A in the diet. This feeding regimen also boosted IgA production and enhanced T follicular helper and mucosal germinal center responses. Mice lacking GPR43 or GPR109A, receptors for SCFAs, showed exacerbated food allergy and fewer CD103(+) DCs. Dietary elements, including fiber and vitamin A, therefore regulate numerous protective pathways in the gastrointestinal tract, necessary for immune non-responsiveness to food antigens. PMID:27332875

  12. Erythromycin, roxithromycin, and clarithromycin: use of slow-binding kinetics to compare their in vitro interaction with a bacterial ribosomal complex active in peptide bond formation.

    Science.gov (United States)

    Dinos, George P; Connell, Sean R; Nierhaus, Knud H; Kalpaxis, Dimitrios L

    2003-03-01

    In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes. PMID:12606769

  13. APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner.

    Science.gov (United States)

    Cun, Yanping; Dai, Nan; Li, Mengxia; Xiong, Chengjie; Zhang, Qinhong; Sui, Jiangdong; Qian, Chengyuan; Wang, Dong

    2014-02-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.

  14. Bovine immunoglobulin/protein isolate binds pro-inflammatory bacterial compounds and prevents immune activation in an intestinal co-culture model.

    Science.gov (United States)

    Detzel, Christopher J; Horgan, Alan; Henderson, Abigail L; Petschow, Bryon W; Warner, Christopher D; Maas, Kenneth J; Weaver, Eric M

    2015-01-01

    Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI

  15. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

  16. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    Science.gov (United States)

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  17. Monoclonal antibodies against accumulation-associated protein affect EPS biosynthesis and enhance bacterial accumulation of Staphylococcus epidermidis.

    Directory of Open Access Journals (Sweden)

    Jian Hu

    Full Text Available Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap that contains sequence repeats known as G5 domains, which are responsible for the Zn(2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5 were generated. MAb(18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb(25C11 and MAb(20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb(18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb(25C11 and MAb(20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.

  18. The phytohormone ethylene enhances bacterial cellulose production, regulates CRP/FNRKx transcription and causes differential gene expression within the cellulose synthesis operon of Komagataeibacter (Gluconacetobacter xylinus ATCC 53582

    Directory of Open Access Journals (Sweden)

    Richard Vincent Augimeri

    2015-12-01

    Full Text Available Komagataeibacter (formerly Gluconacetobacter xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx. Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR, we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA induced differential expression of genes within the bacterial cellulose synthesis (bcs operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature.

  19. The Phytohormone Ethylene Enhances Cellulose Production, Regulates CRP/FNRKx Transcription and Causes Differential Gene Expression within the Bacterial Cellulose Synthesis Operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582.

    Science.gov (United States)

    Augimeri, Richard V; Strap, Janice L

    2015-01-01

    Komagataeibacter (formerly Gluconacetobacter) xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC) biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid) to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx). Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx, and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA) induced differential expression of genes within the bacterial cellulose synthesis (bcs) operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature.

  20. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    Science.gov (United States)

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

  1. The Phytohormone Ethylene Enhances Cellulose Production, Regulates CRP/FNRKx Transcription and Causes Differential Gene Expression within the Bacterial Cellulose Synthesis Operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582.

    Science.gov (United States)

    Augimeri, Richard V; Strap, Janice L

    2015-01-01

    Komagataeibacter (formerly Gluconacetobacter) xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC) biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid) to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx). Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx, and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA) induced differential expression of genes within the bacterial cellulose synthesis (bcs) operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature. PMID:26733991

  2. Bacterial glycosyltransferase toxins.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

  3. Construction and evaluation of an exopolysaccharide-producing engineered bacterial strain by protoplast fusion for microbial enhanced oil recovery.

    Science.gov (United States)

    Sun, Shanshan; Luo, Yijing; Cao, Siyuan; Li, Wenhong; Zhang, Zhongzhi; Jiang, Lingxi; Dong, Hanping; Yu, Li; Wu, Wei-Min

    2013-09-01

    Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests. PMID:23856587

  4. An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Wade, M; Blake, M C; Jambou, R C;

    1995-01-01

    An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular ...

  5. The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors.

    Science.gov (United States)

    Cooper, Stacy; Guo, Hong; Friedman, Alan D

    2015-01-01

    The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation.

  6. Interleukin-1 interaction with neuroregulatory systems: selective enhancement by recombinant human and mouse interleukin-1 of in vitro opioid peptide receptor binding in rat brain

    International Nuclear Information System (INIS)

    Interleukin-1 (IL-1) exerts a wide variety of biological effects on various cell types and may be regarded as a pleiotropic peptide hormone. Biological evidence suggests that IL-1 participates in the modulation of central nervous system physiology and behavior in a fashion characteristic of neuroendocrine hormones. In this investigation, recombinant (r) human (h) IL-1 and r mouse (m) IL-1 were examined for their modulation of opioid peptide receptor binding in vitro. Experiments were performed on frozen sections of rat brain. Receptor binding of radiolabeled substance P and of radiolabeled neurotensin were not significantly affected by the presence of rIL-1s. Recombinant IL-1s, however, significantly enhanced specific binding of 125I-beta-endorphin (125I-beta-END) and of D-ala2-(tyrosyl-3,5-3H)enkephalin-(5-D-leucine) (3H-D-ALA), equipotently and in a concentration-dependent manner with maximal activity occurring at a concentration of 10 LAF units/ml. The increased binding of 125I-beta-END and 3H-D-ALA was blocked steroselectively by (-)-naloxone and by etorphine, suggesting detection of opiate receptors. In addition, brain distribution patterns of receptors labeled in the presence of rIL-1s corresponded to patterns previously published for opiate receptors. Autoradiographic visualization of receptors revealed that rIL-1s in the different areas of the brain exert their effect on opioid binding with comparable potencies. The data suggest that certain central nervous system effects of IL-1s may be mediated by their selective interaction with opiatergic systems at the receptor level

  7. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  8. CCAAT/Enhancer-Binding Protein \\(\\gamma\\) Is a Critical Regulator of IL-1\\(\\beta\\)-Induced IL-6 Production in Alveolar Epithelial Cells

    OpenAIRE

    Chunguang Yan; Ximo Wang; Jay Cao; Min Wu; Hongwei Gao

    2012-01-01

    CCAAT/enhancer binding protein \\(\\gamma\\) (C/EBPγ) is a member of the C/EBP family of transcription factors, which lacks known activation domains. C/EBP\\(\\gamma\\) was originally described as an inhibitor of C/EBP transactivation potential. However, previous study demonstrates that C/EBP\\(\\gamma\\) augments the C/EBP\\(\\beta\\) stimulatory activity in lipopolysaccharide induction of IL-6 promoter in a B lymphoblast cell line. These data indicate a complexing functional role for C/EBP\\(\\gamma\\) in...

  9. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    OpenAIRE

    Price, S R; Nightingale, M.; Tsai, S C; Williamson, K. C.; Adamik, R; H. C. Chen; Moss, J; M. Vaughan

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on th...

  10. Mononuclear phagocyte system depletion blocks interstitial tonicity-responsive enhancer binding protein/vascular endothelial growth factor C expression and induces salt-sensitive hypertension in rats.

    Science.gov (United States)

    Machnik, Agnes; Dahlmann, Anke; Kopp, Christoph; Goss, Jennifer; Wagner, Hubertus; van Rooijen, Nico; Eckardt, Kai-Uwe; Müller, Dominik N; Park, Joon-Keun; Luft, Friedrich C; Kerjaschki, Dontscho; Titze, Jens

    2010-03-01

    We showed recently that mononuclear phagocyte system (MPS) cells provide a buffering mechanism for salt-sensitive hypertension by driving interstitial lymphangiogenesis, modulating interstitial Na(+) clearance, and increasing endothelial NO synthase protein expression in response to very high dietary salt via a tonicity-responsive enhancer binding protein/vascular endothelial growth factor C regulatory mechanism. We now tested whether isotonic saline and deoxycorticosterone acetate (DOCA)-salt treatment leads to a similar regulatory response in Sprague-Dawley rats. Male rats were fed a low-salt diet and received tap water (low-salt diet LSD), 1.0% saline (high-salt diet HSD), or DOCA+1.0% saline (DOCA-HSD). To test the regulatory role of interstitial MPS cells, we further depleted MPS cells with clodronate liposomes. HSD and DOCA-HSD led to Na(+) accumulation in the skin, MPS-driven tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated hyperplasia of interstitial lymph capillaries, and increased endothelial NO synthase protein expression in skin interstitium. Clodronate liposome MPS cell depletion blocked MPS infiltration in the skin interstitium, resulting in unchanged tonicity-responsive enhance binding protein/vascular endothelial growth factor C levels and absent hyperplasia of the lymph capillary network. Moreover, no increased skin endothelial NO synthase protein expression occurred in either clodronate liposome-treated HSD or DOCA-salt rats. Thus, absence of the MPS-cell regulatory response converted a salt-resistant blood-pressure state to a salt-sensitive state in HSD rats. Furthermore, salt-sensitive hypertension in DOCA-salt rats was aggravated. We conclude that MPS cells act as onsite controllers of interstitial volume and blood pressure homeostasis, providing a local regulatory salt-sensitive tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated mechanism in the skin to maintain

  11. Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

    OpenAIRE

    Cho, Eun-Young; Kim, Hyung-Jin; Park, Channy; So, Hong-Seob; Park, Rae Kil; Kim, Haak Cheoul

    2013-01-01

    Background/Aims The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication. Methods We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluat...

  12. Enhanced binding affinity, remarkable selectivity, and high capacity of CO 2 by dual functionalization of a rht-type metal-organic framework

    KAUST Repository

    Li, Baiyan

    2011-12-23

    Open and friendly: The smallest member of the rht-type metal-organic frameworks (MOFs, see picture) constructed by a hexacarboxylate ligand with a nitrogen-rich imino triazine backbone shows a significantly enhanced gas binding affinity relative to all other isoreticular rht-type MOFs. The high adsorption capacity and remarkable selectivity of CO 2 are attributed to the high density of open metal and Lewis basic sites in the framework. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    Science.gov (United States)

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  14. Evanescent-wave cavity ring-down spectroscopy for enhanced detection of surface binding under flow injection analysis conditions.

    Science.gov (United States)

    van der Sneppen, L; Buijs, J B; Gooijer, C; Ubachs, W; Ariese, F

    2008-06-01

    The feasibility of liquid-phase evanescent-wave cavity ring-down spectroscopy (EW-CRDS) for surface-binding studies under flow-injection analysis (FIA) conditions is demonstrated. The EW-CRDS setup consists of an anti-reflection coated Dove prism inside a linear cavity (with standard or super-polishing of the total internal reflective (TIR) surface). A teflon spacer with an elliptical hole clamped on this surface acts as a 20 muL sized flow cell. The baseline noise of this system is of the order of 10(-4) absorbance units; the baseline remains stable over a prolonged time and the prism surface does not become contaminated during repeated injections of the reversibly adsorbing test dyes Crystal Violet (CV) and Direct Red 10 (DR10). At typical FIA or liquid chromatography (LC) flow rates, the system has sufficient specificity to discriminate between species with different surface affinities. For CV a much stronger decrease in ring-down time is observed than calculated based on its bulk concentration and the effective depth probed by the evanescent wave, indicating binding of this positively charged dye to the negatively charged prism surface. The amount of adsorption can be influenced by adjusting the flow rate or the eluent composition. At a flow rate of 0.5 mL/min, an enrichment factor of 60 was calculated for CV; for the poorly adsorbing dye DR10 it is 5. Super-polishing of the already polished TIR surface works counter-productively. The adsorbing dye Crystal Violet has a detection limit of 3 muM for the standard polished surface; less binding occurs on the super-polished surface and the detection limit is 5 muM. Possible applications of EW-CRDS for studying surface binding or the development of bio-assays are discussed. PMID:18559152

  15. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-01

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  16. Leucine zipper like structure in rice WRKY89 enhances its affinity for binding with W box elements

    Institute of Scientific and Technical Information of China (English)

    WANG Haihua; HAO Zhongna; XIE Ke; WU Kunlu; GUO Zejian

    2005-01-01

    WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of OsWRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.

  17. Enhancing the Decolorizing and Degradation Ability of Bacterial Consortium Isolated from Textile Effluent Affected Area and Its Application on Seed Germination

    Directory of Open Access Journals (Sweden)

    Rashid Mahmood

    2015-01-01

    Full Text Available A bacterial consortium BMP1/SDSC/01 consisting of six isolates was isolated from textile effected soil, sludge, and textile effluent from Hudiara drain near Nishat Mills Limited, Ferozepur Road, Lahore, Pakistan. It was selected because of being capable of degrading and detoxifying red, green, black, and yellow textile dyes. The pH and supplements were optimized to enhance the decolorization ability of the selected consortium. The results indicated that decolorizing ability of consortium for the red, green, black, and yellow dyes was higher as compared to individual strains. The consortium was able to decolorize 84%, 84%, 85%, 85%, and 82% of 200 ppm of red, green, black, yellow, and mixed dyes within 24 h while individual strain required 72 h. On supplementing urea, the consortium decolorized 87, 86, 89, 86, and 83%, respectively, while on supplementing sodium chloride the consortium decolorized 93, 94, 93, 94, and 89% of red, green, black, yellow, and mixed dyes, respectively, which was maximum while in the presence of ascorbic acid and ammonium chloride it showed intermediate results. The effect of untreated and treated dyes was investigated on Zea mays L. (maize and Sorghum vulgare Pers. (sorghum. This study will help to promote an efficient biotreatment of textile effluents.

  18. Enhancing the decolorizing and degradation ability of bacterial consortium isolated from textile effluent affected area and its application on seed germination.

    Science.gov (United States)

    Mahmood, Rashid; Sharif, Faiza; Ali, Sikander; Hayyat, Muhammad Umar

    2015-01-01

    A bacterial consortium BMP1/SDSC/01 consisting of six isolates was isolated from textile effected soil, sludge, and textile effluent from Hudiara drain near Nishat Mills Limited, Ferozepur Road, Lahore, Pakistan. It was selected because of being capable of degrading and detoxifying red, green, black, and yellow textile dyes. The pH and supplements were optimized to enhance the decolorization ability of the selected consortium. The results indicated that decolorizing ability of consortium for the red, green, black, and yellow dyes was higher as compared to individual strains. The consortium was able to decolorize 84%, 84%, 85%, 85%, and 82% of 200 ppm of red, green, black, yellow, and mixed dyes within 24 h while individual strain required 72 h. On supplementing urea, the consortium decolorized 87, 86, 89, 86, and 83%, respectively, while on supplementing sodium chloride the consortium decolorized 93, 94, 93, 94, and 89% of red, green, black, yellow, and mixed dyes, respectively, which was maximum while in the presence of ascorbic acid and ammonium chloride it showed intermediate results. The effect of untreated and treated dyes was investigated on Zea mays L. (maize) and Sorghum vulgare Pers. (sorghum). This study will help to promote an efficient biotreatment of textile effluents. PMID:25654132

  19. The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor.

    Science.gov (United States)

    Weaver, Ian C G; Hellstrom, Ian C; Brown, Shelley E; Andrews, Stephen D; Dymov, Sergiy; Diorio, Josie; Zhang, Tie-Yuan; Szyf, Moshe; Meaney, Michael J

    2014-09-26

    Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 17 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 17 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 17 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.

  20. N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA.

    Science.gov (United States)

    Hilimire, Thomas A; Bennett, Ryan P; Stewart, Ryan A; Garcia-Miranda, Pablo; Blume, Alex; Becker, Jordan; Sherer, Nathan; Helms, Eric D; Butcher, Samuel E; Smith, Harold C; Miller, Benjamin L

    2016-01-15

    Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides. PMID:26496521

  1. Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing

    Directory of Open Access Journals (Sweden)

    Xianwei Zhu

    2014-01-01

    Full Text Available We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC52-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF, was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC52-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC52-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC52-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

  2. Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip

    DEFF Research Database (Denmark)

    Rahimov, Fedik; Marazita, Mary L; Visel, Axel;

    2008-01-01

    Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P) is strongly associated with SNPs in IRF6 (interferon regulatory factor 6). Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A ...

  3. Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining

    NARCIS (Netherlands)

    N. Gul; M. Bogels; S. Grewal; A.J. van der Meer; L.B. Rojas; D.M. Fluitsma; M.P. van den Tol; K.A. Hoeben; J. van Marle; H.E. de Vries; R.H.J. Beelen; M. van Egmond

    2011-01-01

    Objective Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) ar

  4. Impairment of the bacterial biofilm stability by triclosan.

    Directory of Open Access Journals (Sweden)

    Helen V Lubarsky

    Full Text Available The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition--isolated from sediments of the Eden Estuary (Scotland, UK--on non-cohesive glass beads (<63 µm and exposed to a range of triclosan concentrations (control, 2-100 µg L(-1 was monitored over time by Magnetic Particle Induction (MagPI. In parallel, bacterial cell numbers, division rate, community composition (DGGE and EPS (extracellular polymeric substances: carbohydrates and proteins secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of

  5. Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.

    Science.gov (United States)

    Latinovic, Olga; Schneider, Kate; Szmacinski, Henryk; Lakowicz, Joseph R; Heredia, Alonso; Redfield, Robert R

    2014-12-01

    The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.

  6. Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells.

    Science.gov (United States)

    Duren, Ryan P; Boudreaux, Seth P; Conneely, Orla M

    2016-01-01

    Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells.

  7. Protein-x of hepatitis B virus in interaction with CCAAT/enhancer-binding protein α (C/EBPα - an in silico analysis approach

    Directory of Open Access Journals (Sweden)

    Mohamadkhani Ashraf

    2011-10-01

    Full Text Available Abstract Background Even though many functions of protein-x from the Hepatitis B virus (HBV have been revealed, the nature of protein-x is yet unknown. This protein is well-known for its transactivation activity through interaction with several cellular transcription factors, it is also known as an oncogene. In this work, we have presented computational approaches to design a model to show the structure of protein-x and its respective binding sites associated with the CCAAT/enhancer-binding protein α (C/EBPα. C/EBPα belongs to the bZip family of transcription factors, which activates transcription of several genes through its binding sites in liver and fat cells. The C/EBPα has been shown to bind and modulate enhancer I and the enhancer II/core promoter of HBV. In this study using the bioinformatics tools we tried to present a reliable model for the protein-x interaction with C/EBPα. Results The amino acid sequence of protein-x was extracted from UniProt [UniProt:Q80IU5] and the x-ray crystal structure of the partial CCAAT-enhancer α [PDB:1NWQ] was retrieved from the Protein Data Bank (PDB. Similarity search for protein-x was carried out by psi-blast and bl2seq using NCBI [GenBank: BAC65106.1] and Local Meta-Threading-Server (LOMETS was used as a threading server for determining the maximum tertiary structure similarities. Advanced MODELLER was implemented to design a comparative model, however, due to the lack of a suitable template, Quark was used for ab initio tertiary structure prediction. The PDB-blast search indicated a maximum of 23% sequence identity and 33% similarity with crystal structure of the porcine reproductive and respiratory syndrome virus leader protease Nsp1α [PDB:3IFU]. This meant that protein-x does not have a suitable template to predict its tertiary structure using comparative modeling tools, therefore we used QUARK as an ab initio 3D prediction approach. Docking results from the ab initio tertiary structure of

  8. Enhancement of photophysical and photosensitizing properties of flavin adenine dinucleotide by mutagenesis of the C-terminal extension of a bacterial flavodoxin reductase.

    Science.gov (United States)

    Valle, Lorena; Abatedaga, Inés; Vieyra, Faustino E Morán; Bortolotti, Ana; Cortez, Néstor; Borsarelli, Claudio D

    2015-03-16

    The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified. PMID:25641205

  9. Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells

    OpenAIRE

    Frank Y. Chen; Harris, Linda C.; Joanna S Remack; Brent, Thomas P.

    1997-01-01

    O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found ...

  10. Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

    OpenAIRE

    Il-Hoon Cho; Sung-Min Seo; Jin-Woo Jeon; Se-Hwan Paek

    2009-01-01

    The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab′)2 were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies ...

  11. CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts

    Science.gov (United States)

    Umayahara, Y.; Billiard, J.; Ji, C.; Centrella, M.; McCarthy, T. L.; Rotwein, P.

    1999-01-01

    Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in

  12. Human translocation liposarcoma-CCAAT/enhancer binding protein (C/EBP) homologous protein (TLS-CHOP) oncoprotein prevents adipocyte differentiation by directly interfering with C/EBPbeta function.

    Science.gov (United States)

    Adelmant, G; Gilbert, J D; Freytag, S O

    1998-06-19

    Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process. PMID:9624148

  13. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  14. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    Directory of Open Access Journals (Sweden)

    Sakai Keiko

    2008-04-01

    Full Text Available Abstract Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.

  15. Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood.

    Science.gov (United States)

    Blatt, Adam Z; Saggu, Gurpanna; Kulkarni, Koustubh V; Cortes, Claudio; Thurman, Joshua M; Ricklin, Daniel; Lambris, John D; Valenzuela, Jesus G; Ferreira, Viviana P

    2016-06-01

    Enhanced levels of platelet/granulocyte aggregates (PGAs) are found in patients suffering from many different inflammatory vascular diseases, and their formation in animal models of vascular disease is associated with increased thromboinflammation and worsened outcomes. The complement system, a part of the innate immune system, influences PGA formation, but the mechanisms for its effects are unknown. In this study, we have defined complement-mediated mechanisms that enhance PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP) using ex vivo flow cytometry assays. We demonstrate that physiological properdin, a positive regulator of complement alternative pathway activity, increases PGA formation when added to TRAP-stimulated blood. All physiological properdin forms increase PGA formation, but properdin tetramers are the most efficient at increasing complement activity and PGA formation. Inhibition of endogenous properdin, either circulating in the blood or produced locally by leukocytes, impairs TRAP-mediated PGA formation to the same level as specific inhibition of either the alternative or classical pathway. Additionally, blocking the interaction of C5a with its cellular receptor prevents properdin-mediated increases in PGA formation. Adding either properdin tetramers or C5a to whole blood increases CD11b expression on granulocytes, and this increase is prevented by blockade of the C5a-C5a receptor axis. Finally, we demonstrate that the effects of properdin on PGA formation are tightly regulated by Factor H. Cumulatively, our data indicate that properdin enhances PGA formation via increased production of C5a, and that inhibition of properdin function has therapeutic potential to limit thromboinflammation in diseases characterized by increased PGA formation. PMID:27183616

  16. Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

    Science.gov (United States)

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V; Schuetz, John D

    2013-09-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  17. CCAAT-enhancer-binding Protein β (C/EBPβ) and Downstream Human Placental Growth Hormone Genes Are Targets for Dysregulation in Pregnancies Complicated by Maternal Obesity*

    Science.gov (United States)

    Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A.

    2013-01-01

    Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in “normal” pregnancy. Maternal obesity can exacerbate this “resistance,” suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m2) versus lean (BMI 20–25 kg/m2) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development. PMID:23782703

  18. CCAAT-enhancer-binding protein β (C/EBPβ) and downstream human placental growth hormone genes are targets for dysregulation in pregnancies complicated by maternal obesity.

    Science.gov (United States)

    Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A

    2013-08-01

    Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in "normal" pregnancy. Maternal obesity can exacerbate this "resistance," suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m(2)) versus lean (BMI 20-25 kg/m(2)) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development.

  19. The protein binding substance Ibuprofen does not affect the T1 time or partition coefficient in contrast-enhanced cardiovascular magnetic resonance

    Directory of Open Access Journals (Sweden)

    Kawel Nadine

    2012-10-01

    Full Text Available Abstract Background Contrast enhanced cardiovascular magnetic resonance (CMR with T1 mapping enables quantification of diffuse myocardial fibrosis. Various factors, however, can interfere with T1 measurements. The purpose of the current study was to assess the effect of co-medication with a typical protein binding drug (Ibuprofen on T1 values in vitro and in vivo. Methods 50 vials were prepared with different concentrations of gadobenate dimeglumine, Ibuprofen and human serum albumin in physiologic NaCl solution and imaged at 1.5T with a spin echo sequence at multiple TRs to measure T1 values and calculate relaxivities. 10 volunteers (5 men; 31±6.3 years were imaged at 1.5T. T1 values for myocardium and blood pool were determined for various time points after administration of 0.15mmol/kg gadobenate dimeglumine using a modified look-locker inversion-recovery sequence before and after administration of Ibuprofen over 24 hours. The partition coefficient was calculated as ΔR1myocardium/ΔR1blood, where R1=1/T1. Results In vitro no significant correlation was found between relaxivity and Ibuprofen concentration, neither in absence (r=−0.15, p=0.40 nor in presence of albumin (r=−0.32, p=0.30. In vivo there was no significant difference in post contrast T1 times of myocardium and blood, respectively and also in the partition coefficient between exam 1 and 2 (p>0.05. There was good agreement of the T1 times of myocardium and blood and the partition coefficient, respectively between exam 1 and 2. Conclusions Contrast enhanced T1 mapping is unaffected by co-medication with the protein binding substance Ibuprofen and has an excellent reproducibility.

  20. Bacterial Vaginosis

    Science.gov (United States)

    ... 586. Related Content STDs during Pregnancy Fact Sheet Pregnancy and HIV, Viral Hepatitis, and STD Prevention Pelvic Inflammatory Disease ( ... Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ... STDs See Also Pregnancy Reproductive ...

  1. Bacterial Meningitis

    Science.gov (United States)

    ... Schedules Preteen & Teen Vaccines Meningococcal Disease Sepsis Bacterial Meningitis Recommend on Facebook Tweet Share Compartir On this ... serious disease. Laboratory Methods for the Diagnosis of Meningitis This manual summarizes laboratory methods used to isolate, ...

  2. The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress.

    Science.gov (United States)

    Velasco, J A; Avila, M A; Notario, V

    1999-01-21

    Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts. We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene. Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream. While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus. Similar mechanisms activated cph in other MCA-treated Syrian hamster cells. The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases. In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP. Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation. Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress. PMID:9989819

  3. IL‑6 and IL‑8 enhance factor H binding to the cell membranes.

    Science.gov (United States)

    Popek, Sylwia; Kapka-Skrzypczak, Lucyna; Sawicki, Krzysztof; Wolińska, Ewa; Skrzypczak, Maciej; Czajka, Magdalena

    2016-05-01

    The aim of the present study was to assess the role of interleukin (IL)‑6 and IL‑8 on the expression of fluid‑phase complement inhibitor, factor H (FH), and FH‑like protein 1 (FHL‑1), in the A2780 ovarian carcinoma cell line. This cell line does not normally produce IL‑6, however, is IL‑6 responsive due to the presence of receptor for IL‑6. The presence of FH and FHL‑1 in the cell lysates was confirmed by western blotting. The levels of FH and FHL‑1 in the medium were determined by enzyme‑linked immunosorbent assay. To evaluate gene expression, reverse transcription‑quantitative polymerase chain reaction was performed. The cellular localization of FH and FHL‑1 in ovarian cancer cells was assessed by immunofluorescence. The present study revealed that FH, contrary to FHL‑1, was secreted by ovarian cancer cells, however, this process was independent of IL stimulation. No significant differences were observed in the concentration of FH in the control cells, when compared with the samples treated with IL‑6/IL‑8. The results of western blotting revealed that the protein expression levels of FH and FHL‑1 were not regulated by IL‑6 and IL‑8 in a dose‑dependent manner. Immunofluorescence analysis confirmed that the A2780 ovarian cancer cell line expressed both membrane bound and intracellular forms of FH and FHL‑1. The present data revealed that the A2780 cells expressed and secreted FH protein and are also able to bind FH and FHL‑1. This may influence the efficiency of complement mediated immunotherapy. PMID:27035765

  4. The CCAAT/enhancer binding protein (C/EBP δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-β peptide

    Directory of Open Access Journals (Sweden)

    Nilsson Lars NG

    2011-04-01

    Full Text Available Abstract Background The transcription factors CCAAT/enhancer binding proteins (C/EBP α, β and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB. In general, C/EBPα is down-regulated, whereas both C/EBPβ and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-β (Aβ deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aβ deposits with the aim of defining new therapeutic targets. Methods Here we have investigated the effects of Aβ on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aβ deposits (tg-ArcSwe by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay. Results We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aβ inhibit up-regulation of C/EBPδ expression in interleukin-1β-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPβ was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aβ deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1β-induced C/EBPδ DNA

  5. TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, “ligand passing”, or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFc-induced cytotoxicity, Hep-2 cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFα-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in Hep-2 cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of Hep-2 cells to hTNFα which especiαlly required TRAF2 binding site. hTR75 could not only mediate partial hTNFα-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity.

  6. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    Science.gov (United States)

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling. PMID:26900020

  7. A Zinc Finger Transcription Factor, αA-Crystallin Binding Protein 1, Is a Negative Regulator of the Chondrocyte-Specific Enhancer of the α1(II) Collagen Gene

    OpenAIRE

    Tanaka, Kazuhiro; Matsumoto, Yoshihiro; Nakatani, Fumihiko; Iwamoto, Yukihide; Yamada, Yoshihiko

    2000-01-01

    Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, αA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the ...

  8. A Polymorphic Enhancer near GREM1 Influences Bowel Cancer Risk through Differential CDX2 and TCF7L2 Binding

    Directory of Open Access Journals (Sweden)

    Annabelle Lewis

    2014-08-01

    Full Text Available A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC. The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in ApcMin mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

  9. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  10. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  11. 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.

  12. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  13. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, mea

  14. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Takayo; Yoshida, Yuichi [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Imai, Yasuharu [Department of Gastroenterology, Ikeda Municipal Hospital, Ikeda, Osaka (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University, Graduate School of Medicine and Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center (ProMRes), Ehime University, Shitsukawa, Toon, Ehime (Japan); Iwamoto, Ryo; Mekada, Eisuke [Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Takehara, Tetsuo [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan)

    2013-07-26

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

  15. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    International Nuclear Information System (INIS)

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis

  16. The mild phenotype in severe hemophilia A with Arg1781His mutation is associated with enhanced binding affinity of factor VIII for factor X.

    Science.gov (United States)

    Yada, Koji; Nogami, Keiji; Wakabayashi, Hironao; Fay, Philip J; Shima, Midori

    2013-06-01

    The clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patient's whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5-10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5-10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ~5 IU/dl. In addition, the R1781H mutant demonstrated an ~1.9-fold decrease in Km for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype. PMID:23467620

  17. Bigelovii A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Blocking NF-κB and CCAAT/Enhancer-Binding Protein δ Pathways

    Directory of Open Access Journals (Sweden)

    Chunguang Yan

    2016-01-01

    Full Text Available Optimal methods are applied to acute lung injury (ALI and the acute respiratory distress syndrome (ARDS, but the mortality rate is still high. Accordingly, further studies dedicated to identify novel therapeutic approaches to ALI are urgently needed. Bigelovii A is a new natural product and may exhibit anti-inflammatory activity. Therefore, we sought to investigate its effect on lipopolysaccharide- (LPS- induced ALI and the underlying mechanisms. We found that LPS-induced ALI was significantly alleviated by Bigelovii A treatment, characterized by reduction of proinflammatory mediator production, neutrophil infiltration, and lung permeability. Furthermore, Bigelovii A also downregulated LPS-stimulated inflammatory mediator expressions in vitro. Moreover, both NF-κB and CCAAT/enhancer-binding protein δ (C/EBPδ activation were obviously attenuated by Bigelovii A treatment. Additionally, phosphorylation of both p38 MAPK and ERK1/2 (upstream signals of C/EBPδ activation in response to LPS challenge was also inhibited by Bigelovii A. Therefore, Bigelovii A could attenuate LPS-induced inflammation by suppression of NF-κB, inflammatory mediators, and p38 MAPK/ERK1/2—C/EBPδ, inflammatory mediators signaling pathways, which provide a novel theoretical basis for the possible application of Bigelovii A in clinic.

  18. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    Science.gov (United States)

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

  19. Molecular cloning and expression of EgTCTP, encoding a calcium binding protein, enhances the growth of callus in oil palm (Elaeis guineensis, Jacq

    Directory of Open Access Journals (Sweden)

    Alisa Nakkaew

    2010-12-01

    Full Text Available The translationally controlled tumor protein (TCTP has now been identified in evolutionarily diverse organisms and isthought to play an important role in cell growth and cell division. We have identified an EgTCTP gene from Elaeis guineensisJacq. It is a putative protein of 168 amino acids with a calculated molecular mass of 19.2 kDa. EgTCTP has a high homology(84% - 91% identity at the amino acid level to other plant TCTPs from Hevea brasiliensis, Arachis hypogaea and Glycinemax. The recombinant EgTCTP protein is a calcium binding protein. Transgenic embryonic calli overexpressing EgTCTP havea faster growth rate than non-transformed and empty vector transformed calli. The results show that the enhancement ofEgTCTP gene expression in oil palm embryogenic calli may result in faster multiplication of the embryogenic calli. EgTCTPacts as another Ca2+-modulated protein that is involved in the cell cycle progression.

  20. Overexpression of PGC‑1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein.

    Science.gov (United States)

    Shin, Sung-Won; Yun, Seong-Hoon; Park, Eun-Seon; Jeong, Jin-Sook; Kwak, Jong-Young; Park, Joo-In

    2015-03-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC‑1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC‑1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC‑1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC‑1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC‑1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC‑1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC‑1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC‑1α (PGC‑1α-HEK293 cells) compared to those expressing an empty vector. In PGC‑1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC‑1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC‑1α may promote cell proliferation and tumorigenesis through upregulation of ACBP

  1. Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery

    OpenAIRE

    Anna V. Piterina; John Bartlett; Tony Pembroke, J.

    2010-01-01

    Molecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sl...

  2. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...... biogeochemical processes are carried exclusively by bacteria. * Bacteria play an important role in all types of habitats including some that cannot support eukaryotic life....

  3. [Bacterial vaginosis].

    Science.gov (United States)

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  4. Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA.

    OpenAIRE

    Moskaluk, C A; Bastia, D

    1988-01-01

    The E2 gene of bovine papillomavirus type 1 positively and negatively regulates the transcriptional enhancer located in the long control region of the viral genome. The DNA-binding domain of the E2 gene product was suspected to interact with the DNA sequence motif ACCN6GGT. We have shown that the carboxy-terminal 126 amino acids of the E2 protein constitute the DNA-binding domain. In this paper we described the expression of the E2 carboxy terminus in Escherichia coli and its subsequent purif...

  5. PEROXOTITANATE- AND MONOSODIUM METAL-TITANATE COMPOUNDS AS INHIBITORS OF BACTERIAL GROWTH

    Energy Technology Data Exchange (ETDEWEB)

    Hobbs, D.

    2011-01-19

    Sodium titanates are ion-exchange materials that effectively bind a variety of metal ions over a wide pH range. Sodium titanates alone have no known adverse biological effects but metal-exchanged titanates (or metal titanates) can deliver metal ions to mammalian cells to alter cell processes in vitro. In this work, we test a hypothesis that metal-titanate compounds inhibit bacterial growth; demonstration of this principle is one prerequisite to developing metal-based, titanate-delivered antibacterial agents. Focusing initially on oral diseases, we exposed five species of oral bacteria to titanates for 24 h, with or without loading of Au(III), Pd(II), Pt(II), and Pt(IV), and measuring bacterial growth in planktonic assays through increases in optical density. In each experiment, bacterial growth was compared with control cultures of titanates or bacteria alone. We observed no suppression of bacterial growth by the sodium titanates alone, but significant (p < 0.05, two-sided t-tests) suppression was observed with metal-titanate compounds, particularly Au(III)-titanates, but with other metal titanates as well. Growth inhibition ranged from 15 to 100% depending on the metal ion and bacterial species involved. Furthermore, in specific cases, the titanates inhibited bacterial growth 5- to 375-fold versus metal ions alone, suggesting that titanates enhanced metal-bacteria interactions. This work supports further development of metal titanates as a novel class of antibacterials.

  6. Differential dimerization of variants linked to enhanced S-Cone Sensitivity Syndrome (ESCS) located in the NR2E3 ligand-binding domain

    Science.gov (United States)

    von Alpen, Désirée; Tran, H. Viet; Guex, Nicolas; Venturini, Giulia; Munier, Francis L.; Schorderet, Daniel F.; Haider, Neena B.; Escher, Pascal

    2016-01-01

    NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of Scones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET2) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function. PMID:25703721

  7. Enhancement of the Regenerative Potential of Anorganic Bovine Bone Graft Utilizing a Polyglutamate-Modified BMP2 Peptide with Improved Binding to Calcium-Containing Materials.

    Science.gov (United States)

    Bain, Jennifer L; Bonvallet, Paul P; Abou-Arraj, Ramzi V; Schupbach, Peter; Reddy, Michael S; Bellis, Susan L

    2015-09-01

    Autogenous bone is the gold standard material for bone grafting in craniofacial and orthopedic regenerative medicine. However, due to complications associated with harvesting donor bone, clinicians often use commercial graft materials that may lose their osteoinductivity due to processing. This study was aimed to functionalize one of these materials, anorganic bovine bone (ABB), with osteoinductive peptides to enhance regenerative capacity. Two peptides known to induce osteoblastic differentiation of mesenchymal stem cells were evaluated: (1) DGEA, an amino acid motif within collagen I and (2) a biomimetic peptide derived from bone morphogenic protein 2 (BMP2pep). To achieve directed coupling of the peptides to the graft surface, the peptides were engineered with a heptaglutamate domain (E7), which confers specific binding to calcium moieties within bone mineral. Peptides with the E7 domain exhibited greater anchoring to ABB than unmodified peptides, and E7 peptides were retained on ABB for at least 8 weeks in vivo. To assess the osteoinductive potential of the peptide-conjugated ABB, ectopic bone formation was evaluated utilizing a rat subcutaneous pouch model. ABB conjugated with full-length recombinant BMP2 (rBMP2) was also implanted as a model for current clinical treatments utilizing rBMP2 passively adsorbed to carriers. These studies showed that E7BMP2pep/ABB samples induced more new bone formation than all other peptides, and an equivalent amount of new bone as compared with rBMP2/ABB. A mandibular defect model was also used to examine intrabony healing of peptide-conjugated ABB. Bone healing was monitored at varying time points by positron emission tomography imaging with (18)F-NaF, and it was found that the E7BMP2pep/ABB group had greater bone metabolic activity than all other groups, including rBMP2/ABB. Importantly, animals implanted with rBMP2/ABB exhibited complications, including inflammation and formation of cataract-like lesions in the eye, whereas

  8. Glycosylation of Thermomyces lanuginosa lipase enhances surface binding towards phospholipids, but does not significantly influence the catalytic activity

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Svendsen, Allan; Langbjerg, H.;

    2002-01-01

    or PG matrix than Tll. Addition of the substrate analog benzene boronic acid (BBA) increases the binding affinity of the S146A and N33Q mutants, while only small changes are observed for Tll suggesting that the dynamics of the active-site lid influences the binding affinity and that the flexibility...

  9. Synthesis and molecular recognition of novel oligo(ethylenediamino) bridged bis(beta-cyclodextrin)s and their copper(II) complexes: enhanced molecular binding ability and selectivity by multiple recognition.

    Science.gov (United States)

    Liu, Y; You, C C; Li, B

    2001-03-16

    Four bridged bis(beta-cyclodextrin)s tethered by different lengths of oligo(ethylenediamine)s have been synthesized and their inclusion complexation behavior with selected substrates elucidated by circular dichroism spectroscopy and fluorescence decay. In order to study their binding ability quantitatively, inclusion complexation stability constants with four dye guests, that is, brilliant green (BG), methyl orange (MO), ammonium 8-anilino-1-naphthalenesulfonic acid (ANS), and sodium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), have been determined in aqueous solution at 25 degrees C with spectrophotometric, spectropolarimetric, or spectrofluorometric titrations. The results obtained indicate that the two tethered cyclodextrin units might cooperatively bind to a guest, and the molecular binding ability toward model substrates, especially linear guests such as TNS and MO, could be extended. The tether length plays a crucial role in the molecular recognition, the binding constants for ANS and TNS decrease linearly with an increase in the tether length of dimeric cyclodextrin. The Gibbs free energy changes (-deltaGo) for the unit increment per ethylene are 0.99 kJ mol(-1) for ANS and 0.44 kJmol(-1) for TNS, respectively. On the other hand, the presence of a copper(II) ion in metallobis(beta-cyclodextrin)s oligo(ethylenediamino) tethers enhances not only the original binding ability, but also the molecular selectivity through triple or multiple recognition, as compared with the parent bis(beta-cyclodextrin)s.

  10. The κB transcriptional enhancer motif and signal sequences of V(DJ recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study

    Directory of Open Access Journals (Sweden)

    Wu Lai-Chu

    2002-08-01

    Full Text Available Abstract Background The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N and the carboxyl-DNA binding domain (ZAS-C of a representative family member, named κB DNA binding and recognition component (KRC, were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains. Results Both fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(DJ recombination signal sequences (RSS from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS. Conclusions The RSS are cis-acting DNA motifs which are essential for V(DJ recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(DJ recombination.

  11. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    OpenAIRE

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DN...

  12. Rho-modifying bacterial protein toxins.

    Science.gov (United States)

    Aktories, Klaus

    2015-12-01

    Rho proteins are targets of numerous bacterial protein toxins, which manipulate the GTP-binding proteins by covalent modifications, including ADP ribosylation, glycosylation, adenylylation, proteolytic cleavage and deamidation. Bacterial toxins are important virulence factors but are also potent and efficient pharmacological tools to study the physiological functions of their eukaryotic targets. Recent studies indicate that amazing variations exist in the molecular mechanisms by which toxins attack Rho proteins, which are discussed here.

  13. Bacterial Hydrodynamics

    Science.gov (United States)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  14. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  15. Label-Free Determination of Protein Binding in Aqueous Solution using Overlayer Enhanced Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (OE-ATR-FTIR)

    Science.gov (United States)

    Ruthenburg, Travis; Aweda, Tolulope; Park, Simon; Meares, Claude; Land, Donald

    2009-03-01

    Protein binding/affinity studies are often performed using Surface Plasmon Resonance techniques that don't produce much spectral information. Measurement of protein binding affinity using FTIR is traditionally performed using high protein concentration or deuterated solvent. By immobilizing a protein near the surface of a gold-coated germanium internal reflection element interactions can be measured between an immobilized protein and free proteins or small molecules in aqueous solution. By monitoring the on and off rates of these interactions, the dissociation constant for the system can be determined. The dissociation constant for the molecule Yttrium-DOTA binding to the antibody 2D12.5 system was determined to be 100nM. Results will also be presented from our measurements of Bovine Serum Albumin (BSA) binding to anti-BSA.

  16. Identification and characterization of CCAAT/Enhancer Binding proteindelta (C/EBPdelta target genes in G0 growth arrested mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Huang Tim

    2008-10-01

    Full Text Available Abstract Background CCAAT/Enhancer Binding Proteinδ (C/EBPδ is a member of the highly conserved C/EBP family of leucine zipper (bZIP proteins. C/EBPδ is highly expressed in G0 growth arrested mammary epithelial cells (MECs and "loss of function" alterations in C/EBPδ have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPδ expression has also been reported in breast cancer and acute myeloid leukemia (AML. C/EBPδ functions as a transcriptional activator, however, only a limited number of C/EBPδ target genes have been reported. As a result, the role of C/EBPδ in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPδ contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPδ target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K Array gene chip ("ChIP-chip" assay and to assess the expression and potential functional roles of C/EBPδ target genes in growth control. Results ChIP-chip assays identified ~100 C/EBPδ target gene loci which were classified by gene ontology (GO into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPδ target loci were bound by C/EBPδ in G0 growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPδ-inducible expression of 14/14 C/EBPδ target genes in G0 growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPδ and selected C/EBPδ target genes was also demonstrated in contact-inhibited G0 growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPδ in growth arrested human and murine MECs. Conclusion

  17. Spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Anastasios Koulaouzidis; Shivaram Bhat; Athar A Saeed

    2009-01-01

    Since its initial description in 1964, research has transformed spontaneous bacterial peritonitis (SBP) from a feared disease (with reported mortality of 90%) to a treatable complication of decompensated cirrhosis,albeit with steady prevalence and a high recurrence rate. Bacterial translocation, the key mechanism in the pathogenesis of SBP, is only possible because of the concurrent failure of defensive mechanisms in cirrhosis.Variants of SBP should be treated. Leucocyte esterase reagent strips have managed to shorten the 'tap-toshot' time, while future studies should look into their combined use with ascitic fluid pH. Third generation cephalosporins are the antibiotic of choice because they have a number of advantages. Renal dysfunction has been shown to be an independent predictor of mortality in patients with SBP. Albumin is felt to reduce the risk of renal impairment by improving effective intravascular volume, and by helping to bind proinflammatory molecules. Following a single episode of SBP, patients should have long-term antibiotic prophylaxis and be considered for liver transplantation.

  18. The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

    Science.gov (United States)

    Hastie, Jessica L; Williams, Kyle B; Bohr, Lindsey L; Houtman, Jon C; Gakhar, Lokesh; Ellermeier, Craig D

    2016-09-01

    σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme. This activation requires the proteolytic destruction of the anti-σ factor RsiV via a process of regulated intramembrane proteolysis (RIP). In many cases proteases that cleave at site-1 are thought to directly sense a signal and initiate the RIP process. We previously suggested binding of lysozyme to RsiV initiated the proteolytic destruction of RsiV and activation of σV. Here we determined the X-ray crystal structure of the RsiV-lysozyme complex at 2.3 Å which revealed that RsiV and lysozyme make extensive contacts. We constructed RsiV mutants with altered abilities to bind lysozyme. We find that mutants that are unable to bind lysozyme block site-1 cleavage of RsiV and σV activation in response to lysozyme. Taken together these data demonstrate that RsiV is a receptor for lysozyme and binding of RsiV to lysozyme is required for σV activation. In addition, the co-structure revealed that RsiV binds to the lysozyme active site pocket. We provide evidence that in addition to acting as a sensor for the presence of lysozyme, RsiV also inhibits lysozyme activity. Thus we have demonstrated that RsiV is a protein with multiple functions. RsiV inhibits σV activity in the absence of lysozyme, RsiV binds lysozyme triggering σV activation and RsiV inhibits the enzymatic activity of lysozyme. PMID:27602573

  19. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    Directory of Open Access Journals (Sweden)

    Fauziah Abu Bakar

    2016-04-01

    Full Text Available Bacterial toxin-antitoxin (TA systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.

  20. Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD: Pitfalls and Improved Methodology to Enhance Diversity Recovery

    Directory of Open Access Journals (Sweden)

    Anna V. Piterina

    2010-03-01

    Full Text Available Molecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD, was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments.

  1. The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer.

    OpenAIRE

    Hawley-Nelson, P; Androphy, E J; Lowy, D R; Schiller, J T

    1988-01-01

    The upstream regulatory region (URR) of the bovine papillomavirus (BPV) genome contains an enhancer that is activated by a BPV E2 gene product. We have previously found that a bacterially derived E2 fusion protein specifically interacted with several fragments of URR DNA, suggesting that E2 may activate transcription by directly binding to the enhancer. Each of the bound fragments contains at least one copy of a conserved motif (ACCN6GGT). To determine if this motif is required and sufficient...

  2. Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand.

    Science.gov (United States)

    Parreira, P; Shi, Q; Magalhaes, A; Reis, C A; Bugaytsova, J; Borén, T; Leckband, D; Martins, M C L

    2014-12-01

    The strength of binding between the Helicobacter pylori blood group antigen-binding adhesin (BabA) and its cognate glycan receptor, the Lewis b blood group antigen (Le(b)), was measured by means of atomic force microscopy. High-resolution measurements of rupture forces between single receptor-ligand pairs were performed between the purified BabA and immobilized Le(b) structures on self-assembled monolayers. Dynamic force spectroscopy revealed two similar but statistically different bond populations. These findings suggest that the BabA may form different adhesive attachments to the gastric mucosa in ways that enhance the efficiency and stability of bacterial adhesion.

  3. CCAAT/Enhancer-Binding Protein α Is a Crucial Regulator of Human Fat Mass and Obesity Associated Gene Transcription and Expression

    Directory of Open Access Journals (Sweden)

    Wei Ren

    2014-01-01

    Full Text Available Several susceptibility loci have been reported associated with obesity and T2DM in GWAS. Fat mass and obesity associated gene (FTO is the first gene associated with body mass index (BMI and risk for diabetes in diverse patient populations. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. While much is known about the epigenetic mutations contributing to obesity and T2DM, less is certain with the expression regulation of FTO gene. In this study, a highly conserved canonical C/EBPα binding site was located around position −45~−54 bp relative to the human FTO gene transcriptional start site. Site-directed mutagenesis of the putative C/EBPα binding sites decreased FTO promoter activity. Overexpression and RNAi studies also indicated that C/EBPα was required for the expression of FTO. Chromatin immunoprecipitation (ChIP experiment was carried out and the result shows direct binding of C/EBPα to the putative binding regions in the FTO promoter. Collectively, our data suggest that C/EBPα may act as a positive regulator binding to FTO promoter and consequently, activates the gene transcription.

  4. Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting celluloytic complexes

    NARCIS (Netherlands)

    Mingardon, F.; Chanal, A.; Lopez Contreras, A.M.; Dray, C.; Bayer, E.A.; Fierobe, H.P.

    2007-01-01

    Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostrid

  5. CXCR3 expression defines a novel subset of innate CD8+ T cells that enhance immunity against bacterial infection and cancer upon stimulation with IL-15.

    Science.gov (United States)

    Oghumu, Steve; Terrazas, Cesar A; Varikuti, Sanjay; Kimble, Jennifer; Vadia, Stephen; Yu, Lianbo; Seveau, Stephanie; Satoskar, Abhay R

    2015-03-01

    Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from conventional CD8(+) T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C-X-C motif) receptor 3 (CXCR3)-positive innate CD8(+) T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL-15 activated CXCR3(+) innate CD8(+) T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN-γ(-/-) mice compared with similarly activated CXCR3(-) subset. This was associated with enhanced proliferation and IFN-γ production in CXCR3(+) cells. Further, CXCR3(+) innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3(+) subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a, IL-2Rβ, Atp5e, and Ly6c but reduced IFN-γR2 and Art2b. Ingenuity pathway analysis revealed an up-regulation of genes associated with T-cell activation, proliferation, cytotoxicity, and translational initiation in CXCR3(+) populations. Our results demonstrate that CXCR3 expression in innate CD8(+) T cells defines a subset with enhanced cytotoxic potential and protective antibacterial immune functions. Immunotherapeutic approaches against infectious disease and cancer could utilize CXCR3(+) innate CD8(+) T-cell populations as novel clinical intervention strategies. PMID:25466888

  6. A novel insulin receptor-binding protein from Momordica charantia enhances glucose uptake and glucose clearance in vitro and in vivo through triggering insulin receptor signaling pathway.

    Science.gov (United States)

    Lo, Hsin-Yi; Ho, Tin-Yun; Li, Chia-Cheng; Chen, Jaw-Chyun; Liu, Jau-Jin; Hsiang, Chien-Yun

    2014-09-10

    Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)-binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17-21, 34-40, and 59-66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice. PMID:25144709

  7. Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.

    OpenAIRE

    Weinstein, R E; Walker, F. J.

    1990-01-01

    The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cof...

  8. Angiotensinogen gene-inducible enhancer-binding protein 1, a member of a new family of large nuclear proteins that recognize nuclear factor kappa B-binding sites through a zinc finger motif.

    OpenAIRE

    Ron, D; Brasier, A R; Habener, J F

    1991-01-01

    Transcriptional activation of the rat angiotensinogen gene during the acute-phase response is dependent on a previously characterized acute-phase response element (APRE) that binds at least two types of nuclear proteins: a cytokine-inducible activity indistinguishable from nuclear factor kappa-B (NF kappa B) and a family of C/EBP-like proteins. We screened a rat liver cDNA expression library with a labeled APRE DNA probe and isolated a single clone that encodes a sequence-specific APRE-bindin...

  9. Surface modification of zirconia with polydopamine to enhance fibroblast response and decrease bacterial activity in vitro: A potential technique for soft tissue engineering applications.

    Science.gov (United States)

    Liu, Mingyue; Zhou, Jianfeng; Yang, Yang; Zheng, Miao; Yang, Jianjun; Tan, Jianguo

    2015-12-01

    The quality of soft-tissue integration plays an important role in the short- and long-term success of dental implants. The aim of the present study was to provide a surface modification approach for zirconia implant abutment materials and to evaluate its influence on fibroblast behavior and oral bacteria adhesion, which are the two main factors influencing the quality of peri-implant soft-tissue seal. In this study, polydopamine (PDA)-coated zirconia was prepared and the surface characteristics were evaluated using scanning electron microscopy, atomic force microscopy, a contact-angle-measuring device, X-ray photoelectron spectroscopy, and Raman spectroscopy. The responses of human gingival fibroblasts (HGFs) to PDA-coated zirconia; i.e., adhesion, proliferation, morphology, protein synthesis, and gene expression, were analyzed. Additionally, the adhesion of Streptococcus gordonii and Streptococcus mutans to zirconia after PDA coating was assessed by scanning electron microscopy and live/dead staining. The material surface analyses suggested the successful coating of PDA onto the zirconia surface. The PDA coating significantly increased cell adhesion and proliferation compared with pristine zirconia. HGFs exhibited a high degree of spreading and secreted a high level of collagen type I on PDA-modified disks. Upregulation of integrin α5, β1, β3 and fibronectin was noted in HGFs cultured on PDA-coated zirconia. The number of adherent bacteria decreased significantly on zirconia after PDA coating. In summary, our result suggest that PDA is able to modify the surface of zirconia, influence HGFs' behavior and reduce bacterial adhesion. Therefore, this surface modification approach holds great potential for improving soft-tissue integration around zirconia abutments in clinical application. PMID:26363269

  10. Surface modification of zirconia with polydopamine to enhance fibroblast response and decrease bacterial activity in vitro: A potential technique for soft tissue engineering applications.

    Science.gov (United States)

    Liu, Mingyue; Zhou, Jianfeng; Yang, Yang; Zheng, Miao; Yang, Jianjun; Tan, Jianguo

    2015-12-01

    The quality of soft-tissue integration plays an important role in the short- and long-term success of dental implants. The aim of the present study was to provide a surface modification approach for zirconia implant abutment materials and to evaluate its influence on fibroblast behavior and oral bacteria adhesion, which are the two main factors influencing the quality of peri-implant soft-tissue seal. In this study, polydopamine (PDA)-coated zirconia was prepared and the surface characteristics were evaluated using scanning electron microscopy, atomic force microscopy, a contact-angle-measuring device, X-ray photoelectron spectroscopy, and Raman spectroscopy. The responses of human gingival fibroblasts (HGFs) to PDA-coated zirconia; i.e., adhesion, proliferation, morphology, protein synthesis, and gene expression, were analyzed. Additionally, the adhesion of Streptococcus gordonii and Streptococcus mutans to zirconia after PDA coating was assessed by scanning electron microscopy and live/dead staining. The material surface analyses suggested the successful coating of PDA onto the zirconia surface. The PDA coating significantly increased cell adhesion and proliferation compared with pristine zirconia. HGFs exhibited a high degree of spreading and secreted a high level of collagen type I on PDA-modified disks. Upregulation of integrin α5, β1, β3 and fibronectin was noted in HGFs cultured on PDA-coated zirconia. The number of adherent bacteria decreased significantly on zirconia after PDA coating. In summary, our result suggest that PDA is able to modify the surface of zirconia, influence HGFs' behavior and reduce bacterial adhesion. Therefore, this surface modification approach holds great potential for improving soft-tissue integration around zirconia abutments in clinical application.

  11. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    Science.gov (United States)

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

  12. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  13. Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium.

    Science.gov (United States)

    Chaturvedi, Ruchi; Archana, G

    2014-06-01

    Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20.

  14. Synergistic action of serine- and metallo-proteases from Fusarium oxysporum f. sp. lycopersici cleaves chitin-binding tomato chitinases, reduces their antifungal activity and enhances fungal virulence

    NARCIS (Netherlands)

    Karimi Jashni, M.; Dols, I.; Iida, Y.; Boeren, S.; Beenen, H.G.; Mehrabi, R.; Collemare, J.; Wit, de P.J.G.M.

    2015-01-01

    As part of their defence strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against

  15. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Science.gov (United States)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  16. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  17. On the Binding Stress-Enhanced Sensitivity of (Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO3) 0.35 (PMN-PT) Piezoelectric Plate Sensor (PEPS)

    Science.gov (United States)

    Wu, Wei

    (Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO 3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) showed enhanced sensitivity in chemical and biological sensing applications which has been attributed to binding-induced crystalline orientation switching in the PMN-PT layer. However, so far there has been no direct demonstration of PEPS crystalline orientation switching upon target-analyte binding. Using biotin and streptavidin binding as a model detection system and by direct X-Ray diffraction observations after analyte binding we have unambiguously demonstrated that switching of the crystalline orientations of the PMN-PT layer indeed occurred. In addition, we have shown that PEPS sensitivity enhancement increased with an increasing transverse electromechanical coupling constant, -k31, of the PMN-PT layer--which is known to correlate with the crystalline orientation switching capability--by increasing the grain size of the PMN-PT layer or by applying a DC bias electric field. Finally, unprecedented high sensitivity of PEPS with high -k31, (i.e., -k31 > 0.3) were illustrated by the aM (10-18 M) sensitivity of in situ DNA hybridization detection without amplification and by the 100 fg/ml (10-13 g/ml) sensitivity of rapid, in situ protein detection in biological fluids such as troponin I detection in serum for early sign of myocardial infarction (heart attack), Her2 detection in serum for cancer treatment and monitoring, Tn antigen and anti-Tn antibody detection in serum for early cancer detection, and Toxins detection in stool for Clostridium difficile infection detection.

  18. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    LENUS (Irish Health Repository)

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  19. Mucosal immunization with the Moraxella catarrhalis porin m35 induces enhanced bacterial clearance from the lung: a possible role for opsonophagocytosis

    Directory of Open Access Journals (Sweden)

    Donna eEaston

    2011-05-01

    Full Text Available Moraxella catarrhalis is a significant cause of respiratory tract infection against which a vaccine is sought. Several outer membrane proteins are currently under investigation as potential vaccine antigens, including the porin M35. We have previously shown that the third external loop of M35 was immunodominant over the remainder of the protein for antibody produced in mice against the refolded recombinant protein. However, as this loop is predicted to fold inside the porin channel we also predicted that it would not be accessible to these antibodies when M35 is expressed on the surface of the bacteria in its native conformation. This study investigated the functional activity of antibodies against M35 and those specific for the loop 3 region of M35 in vitro and in vivo. Antisera from mice immunized with M35 or the loop 3-deletion, M35loop3–, recombinant proteins were not bactericidal but did have enhanced opsonic activity, whereas antibodies raised against the loop 3 peptide were not opsonising indicating that the immunodominant loop 3 of M35 was not accessible to antibody as we had previously predicted. Mucosal immunization with M35, M35 that had an antigenically altered loop 3 (M35(ID78 and M35loop3– enhanced the clearance of M. catarrhalis from the lungs of mice challenged with live M. catarrhalis. The in vivo clearance of bacteria in the mice with the M35-derived protein constructs correlated significantly (p<0.001 with the opsonic activity assessed an in vitro opsonophagocytosis assay. This study has demonstrated that the immunodominat B-cell epitope to loop 3 of the M. catarrhalis outer membrane protein M35 is not associated with immune protection and that M35-specific antibodies are not bactericidal but are opsonising. The opsonising activity correlated with in vivo clearance of the bacteria suggesting that opsonising antibody may be a good correlate of immune protection.

  20. Bacterial chromosome organization and segregation.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T

    2015-01-01

    If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria. Here, drawing on studies of many different organisms, we review the emerging picture of how bacterial chromosomes are structured at multiple length scales, highlighting the functions of various DNA-binding proteins and the impact of physical forces. Additionally, we discuss the spatial dynamics of chromosomes, particularly during their segregation to daughter cells. Although there has been tremendous progress, we also highlight gaps that remain in understanding chromosome organization and segregation. PMID:26566111

  1. Structural insights into inhibition of lipid I production in bacterial cell wall synthesis.

    Science.gov (United States)

    Chung, Ben C; Mashalidis, Ellene H; Tanino, Tetsuya; Kim, Mijung; Matsuda, Akira; Hong, Jiyong; Ichikawa, Satoshi; Lee, Seok-Yong

    2016-05-26

    Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraYAA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraYAA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraYAA. Surprisingly, MD2 does not interact with three acidic residues or the Mg(2+) cofactor required for catalysis, suggesting that MD2 binds to MraYAA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraYAA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO. PMID:27088606

  2. Construction of a novel fusion protein harboring mouse inter- feron γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

    Institute of Scientific and Technical Information of China (English)

    丁炎平; 谭维彦; 胡荣; 陈望秋; 侯云德

    1997-01-01

    A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 108 IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against 125I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in in-hibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malig-nant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ-treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.

  3. Binding of Human Fibrinogen to MRP Enhances Streptococcus suis Survival in Host Blood in a αXβ2 Integrin-dependent Manner.

    Science.gov (United States)

    Pian, Yaya; Li, Xueqin; Zheng, Yuling; Wu, Xiaohong; Yuan, Yuan; Jiang, Yongqiang

    2016-01-01

    The Gram-positive bacterium Streptococcus suis serotype 2 (S. suis 2), an important zoonotic pathogen, induces strong systemic infections in humans; sepsis and meningitis are the most common clinical manifestations and are often accompanied by bacteremia. However, the mechanisms of S. suis 2 survival in human blood are not well understood. In our previous study, we identified muramidase-released protein (MRP), a novel human fibrinogen (hFg)-binding protein (FBP) in S. suis 2 that is an important epidemic infection marker with an unknown mechanism in pathogenesis. The present study demonstrates that the N-terminus of MRP (a.a. 283-721) binds to both the Aα and Bβ chains of the D fragment of hFg. Strikingly, the hFg-MRP interaction improved the survival of S. suis 2 in human blood and led to the aggregation and exhaustion of polymorphonuclear neutrophils (PMNs) via an αXβ2 integrin-dependent mechanism. Other Fg-binding proteins, such as M1 (GAS) and FOG (GGS), also induced PMNs aggregation; however, the mechanisms of these FBP-hFg complexes in the evasion of PMN-mediated innate immunity remain unclear. MRP is conserved across highly virulent strains in Europe and Asia, and these data shed new light on the function of MRP in S. suis pathogenesis. PMID:27231021

  4. A New Alkaline pH-Adjusted Medium Enhances Detection of β-Hemolytic Streptococci by Minimizing Bacterial Interference Due to Streptococcus salivarius

    Science.gov (United States)

    Dierksen, Karen P.; Ragland, Nancy L.; Tagg, John R.

    2000-01-01

    A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing Streptococcus salivarius against β-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of β-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-N,N′-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of S. salivarius bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of β-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The β-hemolytic streptococcal isolates recovered from the samples included 314 group A (S. pyogenes), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded S. pyogenes, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of S. pyogenes isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of S. pyogenes isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07; P < 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient S. pyogenes strains which might otherwise go undetected on SBA and other isolation media. PMID:10655361

  5. Sub-chronic exposure to the insecticide dimethoate induces a proinflammatory status and enhances the neuroinflammatory response to bacterial lypopolysaccharide in the hippocampus and striatum of male mice

    Energy Technology Data Exchange (ETDEWEB)

    Astiz, Mariana, E-mail: marianaastiz@gmail.com; Diz-Chaves, Yolanda, E-mail: ydiz@cajal.csic.es; Garcia-Segura, Luis M., E-mail: lmgs@cajal.csic.es

    2013-10-15

    Dimethoate is an organophosphorus insecticide extensively used in horticulture. Previous studies have shown that the administration of dimethoate to male rats, at a very low dose and during a sub-chronic period, increases the oxidation of lipids and proteins, reduces the levels of antioxidants and impairs mitochondrial function in various brain regions. In this study, we have assessed in C57Bl/6 adult male mice, whether sub-chronic (5 weeks) intoxication with a low dose of dimethoate (1.4 mg/kg) affects the expression of inflammatory molecules and the reactivity of microglia in the hippocampus and striatum under basal conditions and after an immune challenge caused by the systemic administration of lipopolysaccharide. Dimethoate increased mRNA levels of tumor necrosis factor α (TNFα) and interleukin (IL) 6 in the hippocampus, and increased the proportion of Iba1 immunoreactive cells with reactive phenotype in dentate gyrus and striatum. Lipopolysaccharide caused a significant increase in the mRNA levels of IL1β, TNFα, IL6 and interferon-γ-inducible protein 10, and a significant increase in the proportion of microglia with reactive phenotype in the hippocampus and the striatum. Some of the effects of lipopolysaccharide (proportion of Iba1 immunoreactive cells with reactive phenotype and IL6 mRNA levels) were amplified in the animals treated with dimethoate, but only in the striatum. These findings indicate that a sub-chronic period of administration of a low dose of dimethoate, comparable to the levels of the pesticide present as residues in food, causes a proinflammatory status in the brain and enhances the neuroinflammatory response to the lipopolysaccharide challenge with regional specificity. - Highlights: • The dose of pesticide used was comparable to the levels of residues found in food. • Dimethoate administration increased cytokine expression and microglia reactivity. • Hippocampus and striatum were differentially affected by the treatment.

  6. Bacterial ice crystal controlling proteins.

    Science.gov (United States)

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  7. CCAAT/enhancer binding protein β (C/EBPβ isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    André Albergaria

    Full Text Available P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

  8. Structure-based Analysis to Hu-DNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Swinger,K.; Rice, P.

    2007-01-01

    HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

  9. Histone Deacetylase Inhibitors Enhance the Apoptotic Activity of Insulin-Like Growth Factor Binding Protein-3 by Blocking PKC-Induced IGFBP-3 Degradation

    OpenAIRE

    Oh, Seung Hyun; Whang, Young Mi; Min, Hye-Young; Han, Seung Ho; Kang, Ju-Hee; Song, Ki-Hoon; Glisson, Bonnie S.; Kim, Yeul Hong; Lee, Ho-Young

    2012-01-01

    Overexpression of insulin-like growth factor binding protein (IGFBP)-3 induces apoptosis of cancer cells. However, preexisting resistance to IGFBP-3 could limit its antitumor activities. This study characterizes the efficacy and mechanism of the combination of recombinant IGFBP-3 (rIGFBP-3) and HDAC inhibitors to overcome IGFBP-3 resistance in a subset of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells. The effects of the combination of rIGFBP-3 and ...

  10. Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

    Science.gov (United States)

    Xia, Shui-Li; Lei, Jian-Lin; Du, Mingliang; Wang, Yimin; Cong, Xin; Xiang, Guang-Tao; Li, Lian-Feng; Yu, Shenye; Du, Enqi; Liu, Siguo; Sun, Yuan; Qiu, Hua-Ji

    2016-01-01

    Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines. PMID:27301745

  11. Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

    Science.gov (United States)

    Shen, Miaoqing; Bunaciu, Rodica P; Congleton, Johanna; Jensen, Holly A; Sayam, Lavanya G; Varner, Jeffrey D; Yen, Andrew

    2011-12-01

    All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

  12. Loop replacements with gut-binding peptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens (Stål)

    Science.gov (United States)

    Shao, Ensi; Lin, Li; Chen, Chen; Chen, Hanze; Zhuang, Haohan; Wu, Songqing; Sha, Li; Guan, Xiong; Huang, Zhipeng

    2016-01-01

    Bacillus thuringiensis (Bt) Cry toxins have been used widely in pest managements. However, Cry toxins are not effective against sap-sucking insects (Hemiptera), which limits the application of Bt for pest management. In order to extend the insecticidal spectrum of Bt toxins to the rice brown planthopper (BPH), Nilaparvata lugens, we modified Cry1Ab putative receptor binding domains with selected BPH gut-binding peptides (GBPs). Three surface exposed loops in the domain II of Cry1Ab were replaced with two GBPs (P2S and P1Z) respectively. Bioassay results showed that toxicity of modified toxin L2-P2S increased significantly (~9 folds) against BPH nymphs. In addition, damage of midgut cells was observed from the nymphs fed with L2-P2S. Our results indicate that modifying Cry toxins based on the toxin-gut interactions can broaden the insecticidal spectrum of Bt toxin. This method provides another approach for the development of transgenic crops with novel insecticidal activity against hemipteran insects and insect populations resistant to current Bt transgenic crops. PMID:26830331

  13. Loop replacements with gut-binding peptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens (Stål).

    Science.gov (United States)

    Shao, Ensi; Lin, Li; Chen, Chen; Chen, Hanze; Zhuang, Haohan; Wu, Songqing; Sha, Li; Guan, Xiong; Huang, Zhipeng

    2016-01-01

    Bacillus thuringiensis (Bt) Cry toxins have been used widely in pest managements. However, Cry toxins are not effective against sap-sucking insects (Hemiptera), which limits the application of Bt for pest management. In order to extend the insecticidal spectrum of Bt toxins to the rice brown planthopper (BPH), Nilaparvata lugens, we modified Cry1Ab putative receptor binding domains with selected BPH gut-binding peptides (GBPs). Three surface exposed loops in the domain II of Cry1Ab were replaced with two GBPs (P2S and P1Z) respectively. Bioassay results showed that toxicity of modified toxin L2-P2S increased significantly (~9 folds) against BPH nymphs. In addition, damage of midgut cells was observed from the nymphs fed with L2-P2S. Our results indicate that modifying Cry toxins based on the toxin-gut interactions can broaden the insecticidal spectrum of Bt toxin. This method provides another approach for the development of transgenic crops with novel insecticidal activity against hemipteran insects and insect populations resistant to current Bt transgenic crops. PMID:26830331

  14. Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

    Indian Academy of Sciences (India)

    Girdhar K Pandey; Amita Pandey; Vanga Siva Reddy; Renu Deswal; Alok Bhattacharya; Kailash C Upadhyaya; Sudhir K Sopory

    2007-03-01

    Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

  15. Functionalization of a Rigid Divalent Ligand for LecA, a Bacterial Adhesion Lectin**

    Science.gov (United States)

    Fu, Ou; Pukin, Aliaksei V; Quarles van Ufford, H C; Kemmink, Johan; de Mol, Nico J; Pieters, Roland J

    2015-01-01

    The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P. aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer–protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC). PMID:26478841

  16. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that ...

  17. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    Science.gov (United States)

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  18. Early infection HIV-1 envelope V1-V2 genotypes do not enhance binding or replication in cells expressing high levels of α4β7 integrin.

    Science.gov (United States)

    Etemad, Behzad; Gonzalez, Oscar A; McDonough, Sean; Pena-Cruz, Victor; Sagar, Manish

    2013-11-01

    It has been postulated that HIV-1 envelope properties, such as shorter and less-glycosylated V1-V2 loops commonly observed among non-subtype B early-transmitted viruses, promote utilization of the gut homing integrin α4β7. This property potentially confers an advantage to some HIV-1 variants early after acquisition. We found that replication-competent recombinant viruses incorporating HIV-1 subtype A compact and less-glycosylated early versus chronic phase V1-V2 loops demonstrated no significant difference in binding to α4β7 high CD8⁺ T cells or replication in α4β7 high CD4⁺ T cells. Integrin α4β7 usage does not select for shorter less-glycosylated envelopes during transmission. PMID:23797693

  19. Booster immunization with a partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA.

    Science.gov (United States)

    Bar-Joseph, M; Filatov, V; Gofman, R; Guang, Y; Hadjinicolis, A; Mawassi, M; Gootwine, E; Weisman, Y; Malkinson, M

    1997-08-01

    Groups of rabbits and young lambs were immunized subcutaneously and intramuscularly with a recombinant citrus tristeza virus (CTV) coat protein (rCTV-CP) antigen. Three weeks after primary immunization the animals were divided into two groups that were boosted either with rCTV-CP or with a partially purified preparation of CTV particles (ppCTV). Twelve and 15 days after the last injection, the animals were bled and the binding capacity of the antisera for CTV detection was examined for capture antibodies by the indirect ELISA. Considerably higher ELISA titers were obtained from animals that were boosted with ppCTV than with rCP. Boosting with partially purified native antigens after priming with recombinant antigens is expected to extend the applicability of the antisera for detecting other structural and non-structural viral antigens by trapping ELISA. PMID:9274814

  20. RNA-binding protein HuD reduces triglyceride production in pancreatic β cells by enhancing the expression of insulin-induced gene 1.

    Science.gov (United States)

    Kim, Chongtae; Lee, Heejin; Kang, Hoin; Shin, Jung Jae; Tak, Hyosun; Kim, Wook; Gorospe, Myriam; Lee, Eun Kyung

    2016-04-01

    Although triglyceride (TG) accumulation in the pancreas leads to β-cell dysfunction and raises the chance to develop metabolic disorders such as type 2 diabetes (T2DM), the molecular mechanisms whereby intracellular TG levels are regulated in pancreatic β cells have not been fully elucidated. Here, we present evidence that the RNA-binding protein HuD regulates TG production in pancreatic β cells. Mouse insulinoma βTC6 cells stably expressing a small hairpin RNA targeting HuD (shHuD) (βTC6-shHuD) contained higher TG levels compared to control cells. Moreover, downregulation of HuD resulted in a decrease in insulin-induced gene 1 (INSIG1) levels but not in the levels of sterol regulatory element-binding protein 1c (SREBP1c), a key transcription factor for lipid production. We identified Insig1 mRNA as a direct target of HuD by using ribonucleoprotein immunoprecipitation (RIP) and biotin pulldown analyses. By associating with the 3'-untranslated region (3'UTR) of Insig1 mRNA, HuD promoted INSIG1 translation; accordingly, HuD downregulation reduced while ectopic HuD expression increased INSIG1 levels. We further observed that HuD downregulation facilitated the nuclear localization of SREBP1c, thereby increasing the transcriptional activity of SREBP1c and the expression of target genes involved in lipogenesis; likewise, we observed lower INSIG1 levels in the pancreatic islets of HuD-null mice. Taken together, our results indicate that HuD functions as a novel repressor of lipid synthesis in pancreatic β cells.

  1. The binding of Aβ1-42 to lipid rafts of RBC is enhanced by dietary docosahexaenoic acid in rats: Implicates to Alzheimer's disease.

    Science.gov (United States)

    Hashimoto, Michio; Hossain, Shahdat; Katakura, Masanori; Al Mamun, Abdullah; Shido, Osamu

    2015-06-01

    Once amyloid β peptides (Aβs) of the Alzheimer's disease build up in blood circulation, they are capable of binding to red blood cell (RBC) and inducing hemolysis of RBC. The mechanisms of the interactions between RBC and Aβ are largely unknown; however, it is very important for the therapeutic target of Aβ-induced hemolysis. In the present study, we investigated whether Aβ1-42 interacts with caveolin-1-containing detergent-resistant membranes (DRMs) of RBC and whether the interaction could be modulated by dietary pre-administration of docosahexaenoic acid (DHA). DHA pre-administration to rats inhibited hemolysis by Aβ1-42. This activity was accompanied by increased DHA levels and membrane fluidity and decreased cholesterol level, lipid peroxidation, and reactive oxygen species in the RBCs of the DHA-pretreated rats, suggesting that the antioxidative property of DHA may rescue RBCs from oxidative damage by Aβ1-42. The level of caveolin-1 was augmented in the DRMs of DHA-pretreated rats. Binding between Aβ1-42 and DRMs of RBC significantly increased in DHA-rats. When fluorescently labeled Aβ1-42 (TAMRA-Aβ1-42) was directly infused into the bloodstream, it again occupied the caveolin-1-containing DRMs of the RBCs from the DHA-rats to a greater extent, indicating that circulating Aβs interact with the caveolin-1-rich lipid rafts of DRMs and the interaction is stronger in the DHA-enriched RBCs. The levels of TAMRA-Aβ1-42 also increased in liver DRMs, whereas it decreased in plasma of DHA-pretreated rats. DHA might help clearance of circulating Aβs by increased lipid raft-dependent degradation pathways and implicate to therapies in Alzheimer's disease.

  2. Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release.

    Science.gov (United States)

    Wang, Lei; Qin, Wanhai; Zhang, Jing; Bao, Chuntong; Zhang, Hu; Che, Yanyi; Sun, Changjiang; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Richard, Paul Langford; Lei, Liancheng

    2016-04-05

    Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

  3. Penicillin-Binding Protein Imaging Probes

    OpenAIRE

    Kocaoglu, Ozden; Carlson, Erin E.

    2013-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  4. Final Report - Molecular Mechanisms of Bacterial Mercury Transformation - UCSF

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Susan M. [UCSF

    2014-04-24

    The bacterial mercury resistance (mer) operon functions in Hg biogeochemistry and bioremediation by converting reactive inorganic Hg(II) and organic [RHg(II)]1+ mercurials to relatively inert monoatomic mercury vapor, Hg(0). Its genes regulate operon expression (MerR, MerD, MerOP), import Hg(II) (MerT, MerP, and MerC), and demethylate (MerB) and reduce (MerA) mercurials. We focus on how these components interact with each other and with the host cell to allow cells to survive and detoxify Hg compounds. Understanding how this ubiquitous detoxification system fits into the biology and ecology of its bacterial host is essential to guide interventions that support and enhance Hg remediation. In the current overall project we focused on two aspects of this system: (1) investigations of the energetics of Hg(II)-ligand binding interactions, and (2) both experimental and computational approaches to investigating the molecular mechanisms of Hg(II) acquisition by MerA and intramolecular transfer of Hg(II) prior to reduction within the MerA enzyme active site. Computational work was led by Prof. Jeremy Smith and took place at the University of Tennessee, while experimental work on MerA was led by Prof. Susan Miller and took place at the University of California San Francisco.

  5. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

  6. Preparation of Biologically Active Arabidopsis Ribosomes and Comparison with Yeast Ribosomes for Binding to a tRNA-Mimic that Enhances Translation of Plant Plus-Strand RNA Viruses

    Directory of Open Access Journals (Sweden)

    Vera Aleksey Stupina

    2013-07-01

    Full Text Available Isolation of biologically active cell components from multicellular eukaryotic organisms often poses difficult challenges such as low yields and inability to retain the integrity and functionality of the purified compound. We previously identified a cap-independent translation enhancer (3’CITE in the 3’UTR of Turnip crinkle virus (TCV that structurally mimics a tRNA and binds to yeast 80S ribosomes and 60S subunits in the P-site. Yeast ribosomes were used for these studies due to the lack of methods for isolation of plant ribosomes with high yields and integrity. To carry out studies with more natural components, a simple and efficient procedure has been developed for the isolation of large quantities of high quality ribosomes and ribosomal subunits from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue. Attempts to isolate high quality ribosomes from wheat germ, bean sprouts and evacuolated protoplasts were unsuccessful. Addition of purified Arabidopsis 80S plant ribosomes to ribosome-depleted wheat germ lysates resulted in a greater than 1200-fold enhancement in in vitro translation of a luciferase reporter construct. The TCV 3’CITE bound to ribosomes with a 3 to 7-fold higher efficiency when using plant 80S ribosomes compared with yeast ribosomes, indicating that this viral translational enhancer is adapted to interact more efficiently with host plant ribosomes.

  7. Microfluidic immunomagnetic separation for enhanced bacterial detection

    DEFF Research Database (Denmark)

    Hoyland, James; Kunstmann-Olsen, Casper; Ahmed, Shakil;

    A combined lab-on-a-chip approach combining immunomagnetic separation (IMS) and flow cytometry was developed for the enrichment and detection of salmonella contamination in food samples. Immunomagnetic beads were immobilized in chips consisting of long fractal meanders while contaminated samples ...... to obtain maximum capturing efficiency. The effects of channel volume, path length and number of bends of microfluidic chip on IMS efficiency were also determined....

  8. Paclitaxel-Loaded Polymeric Micelles Modified with MCF-7 Cell-Specific Phage Protein: Enhanced Binding to Target Cancer Cells and Increased Cytotoxicity

    Science.gov (United States)

    Wang, Tao; Petrenko, Valery A.; Torchilin, Vladimir P.

    2010-01-01

    Polymeric micelles are used as pharmaceutical carriers to increase solubility and bioavailability of poorly water-soluble drugs. Different ligands are used to prepare targeted polymeric micelles. Earlier, we developed the method for use of specific landscape phage fusion coat proteins as targeted delivery ligands and demonstrated the efficiency of this approach with doxorubicin-loaded PEGylated liposomes. Here, we describe a MCF-7 cell-specific micellar formulation self-assembled from the mixture of the micelle-forming amphiphilic polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, MCF-7-specific landscape phage fusion coat protein, and the hydrophobic drug paclitaxel. These micelles demonstrated a very low CMC value and specific binding to target cells. Using an in vitro co-culture model, FACS analysis, and fluorescence microscopy we showed that MCF-7 targeted phage micelles preferential bound to target cells compared to non-target cells. As a result, targeted paclitaxel-loaded phage micelles demonstrated a significantly higher cytotoxicity towards target MCF-7 cells than free drug or non-targeted micelle formulations, but failed to show such a differential toxicity towards non-target C166 cells. Overall, cancer cell-specific phage proteins identified from phage display peptide libraries can serve as targeting ligands (“substitute antibody”) for polymeric micelle-based pharmaceutical preparations. PMID:20518562

  9. The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase

    DEFF Research Database (Denmark)

    Juge, N.; Nøhr, J.; Le Gal-Coëffet, M.-F.;

    2006-01-01

    High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha...... of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced...... in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed...

  10. A calcium-binding protein, rice annexin OsANN1, enhances heat stress tolerance by modulating the production of H2O2.

    Science.gov (United States)

    Qiao, Bei; Zhang, Qian; Liu, Dongliang; Wang, Haiqi; Yin, Jingya; Wang, Rui; He, Mengli; Cui, Meng; Shang, Zhonglin; Wang, Dekai; Zhu, Zhengge

    2015-09-01

    OsANN1 is a member of the annexin protein family in rice. The function of this protein and the mechanisms of its involvement in stress responses and stress tolerance are largely unknown. Here it is reported that OsANN1 confers abiotic stress tolerance by modulating antioxidant accumulation under abiotic stress. OsANN1-knockdown [RNA interference (RNAi)] plants were more sensitive to heat and drought stresses, whereas OsANN1-overexpression (OE) lines showed improved growth with higher expression of OsANN1 under abiotic stress. Overexpression of OsANN1 promoted SOD (superoxide dismutase) and CAT (catalase) activities, which regulate H2O2 content and redox homeostasis, suggesting the existence of a feedback mechanism between OsANN1 and H2O2 production under abiotic stress. Higher expression of OsANN1 can provide overall cellular protection against abiotic stress-induced damage, and a significant accumulation of OsANN1-green fluorescent protein (GFP) signals was found in the cytosol after heat shock treatment. OsANN1 also has calcium-binding and ATPase activities in vitro, indicating that OsANN1 has multiple functions in rice growth. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that OsANN1 interacts with OsCDPK24. This cross-talk may provide additional layers of regulation in the abiotic stress response. PMID:26085678

  11. Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene

    OpenAIRE

    Lécine, P; Algarté, M; Rameil, P; Beadling, C; Bucher, P.; Nabholz, M; Imbert, J

    1997-01-01

    The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides ...

  12. Inhibition of bacterial surface colonization by immobilized silver nanoparticles depends critically on the planktonic bacterial concentration.

    Science.gov (United States)

    Wirth, Stacy M; Bertuccio, Alex J; Cao, Feng; Lowry, Gregory V; Tilton, Robert D

    2016-04-01

    Immobilization of antimicrobial silver nanoparticles (AgNPs) on surfaces has been proposed as a method to inhibit biofouling or as a possible route by which incidental releases of AgNPs may interfere with biofilms in the natural environment or in wastewater treatment. This study addresses the ability of planktonic Pseudomonas fluorescens bacteria to colonize surfaces with pre-adsorbed AgNPs. The ability of the AgNP-coated surfaces to inhibit colonization was controlled by the dissolved silver in the system, with a strong dependence on the initial planktonic cell concentration in the suspension, i.e., a strong inoculum effect. This dependence was attributed to a decrease in dissolved silver ion bioavailability and toxicity caused by its binding to cells and/or cell byproducts. Therefore, when the initial cell concentration was high (∼1×10(7)CFU/mL), an excess of silver binding capacity removed most of the free silver and allowed both planktonic growth and surface colonization directly on the AgNP-coated surface. When the initial cell concentration was low (∼1×10(5)CFU/mL), 100% killing of the planktonic cell inoculum occurred and prevented colonization. When an intermediate initial inoculum concentration (∼1×10(6)CFU/mL) was sufficiently large to prevent 100% killing of planktonic cells, even with 99.97% initial killing, the planktonic population recovered and bacteria colonized the AgNP-coated surface. In some conditions, colonization of AgNP-coated surfaces was enhanced relative to silver-free controls, and the bacteria demonstrated a preferential attachment to AgNP-coated, rather than bare, surface regions. The degree to which the bacterial concentration dictates whether or not surface-immobilized AgNPs can inhibit colonization has significant implications both for the design of antimicrobial surfaces and for the potential environmental impacts of AgNPs. PMID:26771749

  13. Enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein

    Science.gov (United States)

    Wang, Tao; D’Souza, Gerard GM; Bedi, Deepa; Fagbohun, Olusegun A; Potturi, L Prasanna; Papahadjopoulos-Sternberg, Brigitte; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Aim To explore cancer cell-specific phage fusion pVIII coat protein, identified using phage display, for targeted delivery of drug-loaded liposomes to MCF-7 breast cancer cells. Material & methods An 8-mer landscape library f8/8 and a biopanning protocol against MCF-7 cells were used to select a landscape phage protein bearing MCF-7-specific peptide. Size and morphology of doxorubicin-loaded liposomes modified with the tumor-specific phage fusion coat protein (phage–Doxil) were determined by dynamic light scattering and freeze-fraction electron microscopy. Topology of the phage protein in liposomes was examined by western blot. Association of phage–Doxil with MCF-7 cells was evaluated by fluorescence microscopy and fluorescence spectrometry. Selective targeting to MCF-7 was shown by FACS using a coculture model with target and nontarget cells. Phage–Doxil-induced tumor cell killing and apoptosis were confirmed by CellTiter-Blue® Assay and caspase-3/CPP32 fluorometric assay. Results A chimeric phage fusion coat protein specific towards MCF-7 cells, identified from a phage landscape library, was directly incorporated into the liposomal bilayer of doxorubicin-loaded PEGylated liposomes (Doxil®) without additional conjugation with lipophilic moieties. Western blotting confirmed the presence of both targeting peptide and pVIII coat protein in the phage–Doxil, which maintained the liposomal morphology and retained a substantial part of the incorporated drug after phage protein incorporation. The binding activity of the phage fusion pVIII coat protein was retained after incorporation into liposomes, and phage–Doxil strongly and specifically targeted MCF-7 cells, demonstrating significantly increased cytotoxicity towards target cells in vitro. Conclusion We present a novel and straightforward method for making tumor-targeted nanomedicines by anchoring specific phage proteins (substitute antibodies) on their surface. PMID:20528452

  14. 利用细菌表面展示技术构建镉耐受性的重组根瘤菌%Construction of Recombinant Rhizobial Strains with Enhanced Tolerance to Cadmium Using Bacterial Cell Surface Display

    Institute of Scientific and Technical Information of China (English)

    方彩云; 谢福莉; 付玮; 李友国

    2011-01-01

    This study aims to construct recombinant thizobia strains with enhanced tolerance to cadmium by using bacterial cell surface displaying technique, and explore their application in bioremediation of Ca2+ contamintated soil under symbiosis with the host leguminous plants.Two recombinant plasmids, pTNIM and pBLIM, were constructed based on the components of bacterial surface display functional gene InaXN, monkey metallothionein tetramer MT4α and two promoters of PnifH and Plac.By tri-parent matting technique, two recombinant plasmids were transferred into recipient Sinorhizobim fredii HN01.An inducible expression type of recombinant thizobium HNOI (pTNIM) and a constitutive expression type recombinant thizobium HN01 (pBLIM) were obtained, respectively.Under pure culture conditions, the adsorption capacity and tolerance ability of the recombinant and recipient strains to cadmium were comparatively examined, which showed the capacities for both aspects of HN01 (pBLIM) were significantly improved, as such their cadmium-adsorption capacities were increased by over two folds.The results also indicated that the recombinant thizobia demonstrated very good static-adsorption characteristics.Lastly, pot plant experiments showed that the amount of Cd2+ absorbed in root nodules and roots of soybean plants inoculated with HN01(pTNIM) was remarkably higher than that with wild-type strain HN01.Fig 5, Tab 1, Ref 22%通过细菌表面展示功能基因InaXN、猴金属硫蛋白α亚基四聚体MT4α和两种启动子PnifH及Plac元件,分别构建两个重组质粒pTNIM和pBLIM.进一步通过三亲本杂交,将重组质粒分别导入费氏中华根瘤菌HN01,分别构建获得诱导表达型重组菌HN01(pTNIM)和组成表达型重组菌HN01(pBLIM).在培养条件下比较测定重组菌和出发菌对镉的吸附能力和耐受性,结果表明,组成表达型重组菌HN01(pBLIM)在上述两个方面的能力得到明显增强,对镉的吸附富集能力提高了2倍.研究还发

  15. Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance

    Directory of Open Access Journals (Sweden)

    Jennifer R. Brown

    2014-09-01

    Full Text Available Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP. Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

  16. Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis.

    Science.gov (United States)

    Galon, Yael; Nave, Roy; Boyce, Joy M; Nachmias, Dikla; Knight, Marc R; Fromm, Hillel

    2008-03-19

    Calmodulin-binding transcription activator (CAMTA) 3 (also called SR1) is a calmodulin-binding transcription factor in Arabidopsis. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) showed enhanced spontaneous lesions. Transcriptome analysis of both mutants revealed 6 genes with attenuated expression and 99 genes with elevated expression. Of the latter, 32 genes are related to defense against pathogens (e.g. WRKY33, PR1 and chitinase). Propagation of a virulent strain of the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea were attenuated in both mutants. Moreover, both mutants accumulated high levels of H2O2. We suggest that CAMTA3 regulates the expression of a set of genes involved in biotic defense responses.

  17. Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Qiang, Weiguang [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Department of Oncology, The Third Affiliated Hospital, Soochow University, Changzhou (China); Wu, Qinqin [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Department of Radiation Oncology, Changzhou Tumor Hospital, Soochow University, Changzhou (China); Zhou, Fuxiang; Xie, Conghua [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China); Wu, Changping, E-mail: wcpzlk@163.com [Department of Oncology, The Third Affiliated Hospital, Soochow University, Changzhou (China); Zhou, Yunfeng, E-mail: yfzhouwhu@163.com [Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan (China)

    2014-03-07

    Highlights: • Down-regulation of TPP1 shortened telomere length in telomerase-negative cells. • Down-regulation of TPP1 induced cell apoptosis in