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Sample records for m2 protein forms

  1. Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein.

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    Anne-Marie Carola Andersson

    Full Text Available The ectodomain of the matrix 2 protein (M2e of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4 was constructed linking M2e (in its consensus sequence to the rotavirus fragment NSP4(98-135; due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.

  2. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

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    Rangaswamy, Udaya S; O'Flaherty, Brigid M; Speck, Samuel H

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  3. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Directory of Open Access Journals (Sweden)

    Udaya S Rangaswamy

    Full Text Available A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68 infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  4. Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein

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    Andersson, Anne-Marie Carola; Håkansson, Kjell Ove; Jensen, Benjamin Anderschou Holbech

    2012-01-01

    by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP4(98-135); due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2...

  5. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

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    Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

    2014-08-29

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

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    Reimers, Kerstin [Klinik fuer Plastische, Hand-und Wiederherstellungschirurgie, Podbielskistrasse 380, D-30659 Hannover (Germany); Buchholz, Katja [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany); Werchau, Hermann [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany)

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  7. Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.

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    Cai, Hui; Zhang, Yu; Lu, Mijia; Liang, Xueya; Jennings, Ryan; Niewiesk, Stefan; Li, Jianrong

    2016-08-15

    Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus

  8. Machining tools in AISI M2 high-speed steel obtained by spray forming process

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    Jesus, Edilson Rosa Barbosa de.

    2004-01-01

    The aim of the present work was the obtention of AISI M2 high-speed steel by spray forming technique and the material evaluation when used as machining tool. The obtained material was hot rolled at 50% and 72% reduction ratios, and from which it was manufactured inserts for machining tests. The performance of inserts made of the spray formed material was compared to inserts obtained from conventional and powder metallurgy (MP) processed materials. The spray formed material was chemical, physical, mechanical and microstructural characterised. For further characterisation, the materials were submitted to machining tests for performance evaluation under real work condition. The results of material characterisation highlight the potential of the spray forming technique, in the obtention of materials with good characteristics and properties. Under the current processing, hot rolling and heat treatments condition, the analysis of the results of the machining tests revealed a very similar behaviour among the tested materials. Proceeding a criterious analysis of the machining results tests, it was verified that the performance presented by the powder metallurgy material (MP) was slight superior, followed by conventional obtained material (MConv), which presented a insignificant advantage over the spray formed and hot rolled (72% reduction ratio) material. The worst result was encountered for the spray forming and hot rolled (50% reduction ratio) material that presented the highest wear values. (author)

  9. The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

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    Andrea M Siegel

    2008-04-01

    Full Text Available Murine gammaherpesvirus 68 (MHV68 establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV. EBV encodes an interleukin-10 (IL-10 homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25 and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis

  10. The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

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    Siegel, Andrea M; Herskowitz, Jeremy H; Speck, Samuel H

    2008-04-04

    Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a

  11. Form factors of descendant operators: reduction to perturbed M(2,2s+1) models

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    Lashkevich, Michael; Pugai, Yaroslav

    2015-01-01

    In the framework of the algebraic approach to form factors in two-dimensional integrable models of quantum field theory we consider the reduction of the sine-Gordon model to the Φ 13 -perturbation of minimal conformal models of the M(2,2s+1) series. We find in an algebraic form the condition of compatibility of local operators with the reduction. We propose a construction that make it possible to obtain reduction compatible local operators in terms of screening currents. As an application we obtain exact multiparticle form factors for the compatible with the reduction conserved currents T ±2k , Θ ±(2k−2) , which correspond to the spin ±(2k−1) integrals of motion, for any positive integer k. Furthermore, we obtain all form factors of the operators T 2k T −2l , which generalize the famous TT̄ operator. The construction is analytic in the s parameter and, therefore, makes sense in the sine-Gordon theory.

  12. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Zhang Yongfang; Xia Zongqin; Hu Ya'er

    2010-01-01

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  13. Multimeric recombinant M2e protein-based ELISA: a significant improvement in differentiating avian influenza infected chickens from vaccinated ones.

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    Farshid Hadifar

    Full Text Available Killed avian influenza virus (AIV vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006 was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.

  14. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  15. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

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    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  16. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

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    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  17. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

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    Junping Ren

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS. Whether M2-2 regulates the innate immunity in human dendritic cells (DC, an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2 produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT, suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88, an essential adaptor for Toll-like receptors (TLRs, plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  18. NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

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    Wang, Tuo; Cady, Sarah D.; Hong, Mei

    2012-01-01

    The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

  19. Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

    International Nuclear Information System (INIS)

    Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan

    2016-01-01

    The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

  20. Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

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    Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2016-11-15

    The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

  1. Microdomain forming proteins in oncogenesis

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    I. B. Zborovskaya

    2016-01-01

    Full Text Available Lipid rafts are lateral assembles of cholesterol, sphingomyelin, glicosphingolipids and specific proteins within cell plasma membrane. These microdomains are involved into a number of important cellular processes including membrane rearrangement, protein internalization, signal transduction, entry of viruses into the cell. Some of lipid rafts are stabilized by special microdomain-forming proteins such as caveolins, SPFH domain containing superfamily, tetraspanins, galectins, which maintain integrity of rafts and regulate signal transduction via forming of “signalosomes”. Involvement of the different lipid rafts is necessary in many situations such as binding of growth factors with their receptors, integrin regulation, cytoskeleton and extracellular matrix rearrangements, vesicular transport, etc. However, such classes of microdomain-forming proteins are still considered separately from each other. In this review we tried to perform complex analysis of microdomain-forming proteins in regulation of cancer assotiated processes.

  2. Membrane-Dependent Effects of a Cytoplasmic Helix on the Structure and Drug Binding of the Influenza Virus M2 Protein

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    Cady, Sarah; Wang, Tuo; Hong, Mei

    2011-01-01

    The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22 to 46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45 to 62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21–61). 13C-2H distance measurements of 13C-labeled protein and 2H-labeled amantadine showed that in DMPC bilayers, the first equivalent of drug bound S31 inside the M2(21–61) pore, similar to the behavior of M2TM in DMPC bilayers. The non-specific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21–61) is S31 in the TM pore. Interestingly, when M2(21–61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, while M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state, but did not rescue drug binding to M2(21–61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helices to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

  3. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    International Nuclear Information System (INIS)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-01-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

  4. Growth kinetics of boride coatings formed at the surface AISI M2 during dehydrated paste pack boriding

    Energy Technology Data Exchange (ETDEWEB)

    Doñu Ruiz, M.A., E-mail: mdonur0800@alumno.ipn.mx [Universidad Politécnica del Valle de México UPVM, Grupo Ciencia e Ingeniería de Materiales, Av. Mexiquense S/N Esquina Av. Universidad Politécnica, Col Villa Esmeralda, 54910 Tultitlan (Mexico); López Perrusquia, N.; Sánchez Huerta, D. [Universidad Politécnica del Valle de México UPVM, Grupo Ciencia e Ingeniería de Materiales, Av. Mexiquense S/N Esquina Av. Universidad Politécnica, Col Villa Esmeralda, 54910 Tultitlan (Mexico); Torres San Miguel, C.R.; Urriolagoitia Calderón, G.M. [Instituto Politécnico Nacional, SEPI-ESIME, Unidad Profesional Adolfo López Mateos Zacatenco, Edificio 5, 2do. Piso, Col. Lindavista, CP 07738 México, D.F. (Mexico); Cerillo Moreno, E.A. [Universidad Politécnica del Valle de México UPVM, Grupo Ciencia e Ingeniería de Materiales, Av. Mexiquense S/N Esquina Av. Universidad Politécnica, Col Villa Esmeralda, 54910 Tultitlan (Mexico); Cortes Suarez, J.V. [Univerisdad Autónoma Metropolitana Unidad Azcapotzalco, Av. San Pablo 180 Azcapotzalco 02200, Área de Ciencia de los Materiales, México, D.F. (Mexico)

    2015-12-01

    The growth kinetics of the boride coatings (FeB and Fe{sub 2}B) at the surface of AISI M2 high speed steels were studied in this work. Boriding thermochemical treatment was carried out by dehydrated paste pack at three different temperatures 1173, 1223, and 1273 K and four exposure times 1, 3, 5, and 7 h, respectively. The presence of FeB and Fe{sub 2}B phases was identified by scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) and X-ray diffraction method. In order to obtain the boron diffusion coefficients at the FeB/Fe{sub 2}B boride coatings, a mathematical model based on the mass balance at the growing interfaces was proposed under certain assumptions. Likewise the parabolic growth constants and the boride incubation time were established as a function of the parameters η (T) and ε (T). The activation energy values estimated for the FeB and Fe{sub 2}B layers were 233.42 and 211.89 kJ mol{sup −1} respectively. A good agreement was obtained between the simulated values of boride layer thicknesses and the experimental results. Finally, empirical relationships of boride coating thickness as a function of boriding temperature and time are presented. - Highlights: • Formed boride coatings at the surface of AISI M2 high speed steels by new process dehydrated paste pack boriding. • The model was based on the mass balance equation at the FeB/Fe{sub 2}B and Fe{sub 2}B/Fe interfaces by considering the boride incubation time. • A good agreement was obtained between the simulated values of boride layers coatings and the experimental results.

  5. Muscarinic M2 receptors directly activate Gq/11 and Gs G-proteins

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

    2007-01-01

    Roč. 320, č. 2 (2007), s. 607-614 ISSN 0022-3565 R&D Projects: GA ČR GP305/05/P209; GA ČR GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic * siRNA * G-proteins Subject RIV: CE - Biochemistry Impact factor: 4.003, year: 2007

  6. The M2 Channel

    DEFF Research Database (Denmark)

    Santner, Paul

    Drug resistance of Influenza A against antivirals is an increasing problem. No effective Influenza A drugs targeting the crucial viral protein, the proton transporter M2 are available anymore due to widespread resistance. Thanks to research efforts elucidating M2 protein structure, function and i...... resistance escape routes from drug inhibition. We thereby were hopefully able to provide a platform for the large-scale evaluation of M2 channel activity, inhibitors and resistance....

  7. Activation of multiple G-proteins by muscarinic M1 and M2 receptors

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

    2006-01-01

    Roč. 27, č. S1 (2006), s. 404-404 ISSN 1671-4083. [World Congress of Pharmacology /15./. 02.07.2006-07.07.2006, Beijing] R&D Projects: GA ČR(CZ) GP305/05/P209; GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * multiple G-protein coupling Subject RIV: ED - Physiology

  8. Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

    Directory of Open Access Journals (Sweden)

    Helen M Wise

    Full Text Available Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42 with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

  9. Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo.

    Science.gov (United States)

    Cai, Hui; Zhang, Yu; Ma, Yuanmei; Sun, Jing; Liang, Xueya; Li, Jianrong

    2015-06-01

    Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute

  10. Studies on Nanoparticle Based Avian Influenza Vaccines to Present Immunogenic Epitopes of the Virus with Concentration on Ectodomain of Matrix 2 (M2e) Protein

    Science.gov (United States)

    Babapoor Dighaleh, Sankhiros

    2011-12-01

    Avian influenza is an infectious disease of avian species caused by type A influenza viruses with a significant economic impact on the poultry industry. Vaccination is the main prevention strategy in many countries worldwide. However, available vaccines elicit antibodies against two major surface protein of the virus hemagglutinin (HA) and neuraminidase (NA), where they constantly change by point mutations. Influenza viruses can also easily undergo gene reassortment. Therefore, to protect chickens against new strain of avian influenza virus, as well as control and prevent virus spread among farms, new vaccines needed to be designed which is a tedious, time consuming and expensive. Recently, conserved regions of the influenza genome have been evaluated as possible universal vaccines to eliminate constant vaccine updates based on circulating virus. In this study, peptide nanotechnology was used to generate vaccine nanoparticles that carry the highly conserved external domain of matrix 2 protein (M2e). These nanoparticles presented M2e in monomeric or tetrameric forms, designated as PSC-M2e-CH and BNSC-M2eN-CH. respectively. First, to demonstrate immunogenicity of these nanoparticles, we measured anti-M2e antibody in chickens, particularly when a high dose was applied. Prior to vaccination-challenge study, the challenge dose were determined by oculonasal inoculation of 10 6 EID50 or 107.7 EID50 of low pathogenicity AI virus HSN2 followed by measuring cloacal and tracheal virus shedding. A biphasic virus shedding pattern was observed with two peaks of virus shedding at days 4 and 8 for both tracheal and cloacal swabs. The chickens infected with 107.7 EID50 had significant virus shedding as compared with 106 EID50. Based on results of mentioned studies, a vaccination-challenge study was conducted by using 75mug of each vaccine construct per inoculation (with and without adjuvant) and higher dose of virus for challenge. BN5C-M2e-CH with adjuvant significantly reduced the

  11. Machining tools in AISI M2 high-speed steel obtained by spray forming process; Ferramentas de usinagem em aco rapido AISI M2 obtido por conformacao por 'spray'

    Energy Technology Data Exchange (ETDEWEB)

    Jesus, Edilson Rosa Barbosa de. E-mail: erbjesus@usp.br

    2004-07-01

    The aim of the present work was the obtention of AISI M2 high-speed steel by spray forming technique and the material evaluation when used as machining tool. The obtained material was hot rolled at 50% and 72% reduction ratios, and from which it was manufactured inserts for machining tests. The performance of inserts made of the spray formed material was compared to inserts obtained from conventional and powder metallurgy (MP) processed materials. The spray formed material was chemical, physical, mechanical and microstructural characterised. For further characterisation, the materials were submitted to machining tests for performance evaluation under real work condition. The results of material characterisation highlight the potential of the spray forming technique, in the obtention of materials with good characteristics and properties. Under the current processing, hot rolling and heat treatments condition, the analysis of the results of the machining tests revealed a very similar behaviour among the tested materials. Proceeding a criterious analysis of the machining results tests, it was verified that the performance presented by the powder metallurgy material (MP) was slight superior, followed by conventional obtained material (MConv), which presented a insignificant advantage over the spray formed and hot rolled (72% reduction ratio) material. The worst result was encountered for the spray forming and hot rolled (50% reduction ratio) material that presented the highest wear values. (author)

  12. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

    Science.gov (United States)

    Asenjo, Ana; Villanueva, Nieves

    2016-01-04

    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

    Energy Technology Data Exchange (ETDEWEB)

    Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others

    1994-09-01

    Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

  14. Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.

    Directory of Open Access Journals (Sweden)

    Marie-Lise Blondot

    Full Text Available Respiratory syncytial virus (RSV protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

  15. Avian metapneumovirus M2:2 protein inhibits replication in Vero cells: modification facilitates live vaccine development.

    Science.gov (United States)

    Clubbe, Jayne; Naylor, Clive J

    2011-11-28

    Throughout the world, avian metapneumovirus (AMPV) infection of subtype A is principally controlled by two live vaccines both derived from UK field strain #8544. Improvements of those vaccines by use of reverse genetics technology was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based reverse genetics system. A systematic reverse genetics based genome modification of a DNA copy of #8544, employing sequence data from a Vero grown, #8544 derived, live vaccine; was used to determine mutations required to facilitate virus recovery and replication in Vero cells. This identified a single coding substitution in the M2:2 reading frame as responsible. Furthermore, ablation of M2:2 was found to elicit the same outcome. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The study shows that M2:2 modification of field virus #8544 will enable research leading to improved vaccines. This may have more general application to other AMPV field strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

    NARCIS (Netherlands)

    Forrest, LR; Tieleman, DP; Sansom, MSP

    Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within,a membrane protein sequence. However, these methods

  17. Analysis of coupling of M2 muscarinic acetylcholine receptors to Gi/o, Gs and Gq heterotrimeric GTP-binding proteins

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Doležal, Vladimír

    2006-01-01

    Roč. 27, č. S1 (2006), s. 361-361 ISSN 1671-4083. [World Congress of Pharmacology /15./. 02.07.2006-07.07.2006, Beijing] R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : M2 receptor * heterotrimeric G-proteins * analysis of coupling Subject RIV: ED - Physiology

  18. Sequence variation in the M2 ion channel protein influences its intracellular localization during avian influenza replication

    Science.gov (United States)

    Avian influenza virus (AIV) is a constant threat to poultry worldwide due to its ability to mutate from a low pathogenic (LP) form into a highly pathogenic (HP) one. It is known that the incorporation of a polybasic cleavage site (PBCS) on the hemagglutinin (HA) gene is a major indicator of pathogen...

  19. Murine gammaherpesvirus M2 protein induction of IRF4 via the NFAT pathway leads to IL-10 expression in B cells.

    Directory of Open Access Journals (Sweden)

    Udaya S Rangaswamy

    2014-01-01

    Full Text Available Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV, Kaposi's sarcoma-associated herpesvirus (KSHV and murine gammaherpesvirus 68 (MHV68 from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner--leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4. Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- -mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4--a key player in plasma cell differentiation--which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.

  20. Endosulfan induced alteration in bacterial protein profile and RNA yield of Klebsiella sp. M3, Achromobacter sp. M6, and Rhodococcus sp. M2.

    Science.gov (United States)

    Singh, Madhu; Singh, Dileep Kumar

    2014-01-30

    Three bacterial strains identified as Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2 were isolated by soil enrichment with endosulfan followed by shake flask enrichment technique. They were efficiently degrading endosulfan in the NSM (non sulfur medium) broth. Degradation of endosulfan was faster with the cell free extract of bacterial cells grown in the sulfur deficient medium (NSM) supplemented with endosulfan than that of nutrient rich medium (Luria Bertani). In the cell free extract of NSM supplemented with endosulfan as sole sulfur source, a unique band was visualized on SDS-PAGE but not with magnesium sulfate as the sole sulfur source in NSM and LB with endosulfan. Expression of a unique polypeptide band was speculated to be induced by endosulfan under sulfur starved condition. These unique polypeptide bands were identified as OmpK35 protein, sulfate binding protein and outer membrane porin protein, respectively, in Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2. Endosulfan showed dose dependent negative effect on total RNA yield of bacterial strains in nutrient rich medium. Absence of plasmid DNA indicated the presence of endosulfan metabolizing gene on genomic DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex.

    Science.gov (United States)

    Kiss, Gabriella; Holl, Jens M; Williams, Grant M; Alonas, Eric; Vanover, Daryll; Lifland, Aaron W; Gudheti, Manasa; Guerrero-Ferreira, Ricardo C; Nair, Vinod; Yi, Hong; Graham, Barney S; Santangelo, Philip J; Wright, Elizabeth R

    2014-07-01

    Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles

  2. Homogeneous M2 duals

    International Nuclear Information System (INIS)

    Figueroa-O’Farrill, José; Ungureanu, Mara

    2016-01-01

    Motivated by the search for new gravity duals to M2 branes with N>4 supersymmetry — equivalently, M-theory backgrounds with Killing superalgebra osp(N|4) for N>4 — we classify (except for a small gap) homogeneous M-theory backgrounds with symmetry Lie algebra so(n)⊕so(3,2) for n=5,6,7. We find that there are no new backgrounds with n=6,7 but we do find a number of new (to us) backgrounds with n=5. All backgrounds are metrically products of the form AdS 4 ×P 7 , with P riemannian and homogeneous under the action of SO(5), or S 4 ×Q 7 with Q lorentzian and homogeneous under the action of SO(3,2). At least one of the new backgrounds is supersymmetric (albeit with only N=2) and we show that it can be constructed from a supersymmetric Freund-Rubin background via a Wick rotation. Two of the new backgrounds have only been approximated numerically.

  3. Homogeneous M2 duals

    Energy Technology Data Exchange (ETDEWEB)

    Figueroa-O’Farrill, José [School of Mathematics and Maxwell Institute for Mathematical Sciences,The University of Edinburgh,James Clerk Maxwell Building, The King’s Buildings, Peter Guthrie Tait Road,Edinburgh EH9 3FD, Scotland (United Kingdom); Ungureanu, Mara [Humboldt-Universität zu Berlin, Institut für Mathematik,Unter den Linden 6, 10099 Berlin (Germany)

    2016-01-25

    Motivated by the search for new gravity duals to M2 branes with N>4 supersymmetry — equivalently, M-theory backgrounds with Killing superalgebra osp(N|4) for N>4 — we classify (except for a small gap) homogeneous M-theory backgrounds with symmetry Lie algebra so(n)⊕so(3,2) for n=5,6,7. We find that there are no new backgrounds with n=6,7 but we do find a number of new (to us) backgrounds with n=5. All backgrounds are metrically products of the form AdS{sub 4}×P{sup 7}, with P riemannian and homogeneous under the action of SO(5), or S{sup 4}×Q{sup 7} with Q lorentzian and homogeneous under the action of SO(3,2). At least one of the new backgrounds is supersymmetric (albeit with only N=2) and we show that it can be constructed from a supersymmetric Freund-Rubin background via a Wick rotation. Two of the new backgrounds have only been approximated numerically.

  4. 15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

    Science.gov (United States)

    Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

    2005-04-01

    This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

  5. Were protein internal repeats formed by "bricolage"?

    Science.gov (United States)

    Lavorgna, G; Patthy, L; Boncinelli, E

    2001-03-01

    Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

  6. Heterotic M2-branes

    Directory of Open Access Journals (Sweden)

    Neil Lambert

    2015-10-01

    Full Text Available We construct the action for N M2-branes on S1/Z2. The resulting theory has a gauge anomaly but this can be cancelled if the two fixed point planes each support 8 chiral Fermions in the fundamental of U(N. Taking the low energy limit leads to the worldsheet theory of N free heterotic strings whose quantization induces an E8 spacetime gauge symmetry on each fixed point plane. Thus this paper presents a non-abelian worldvolume analogue of the classic Hořava–Witten analysis.

  7. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Scherer, N.M.; Nathanson, N.M.

    1990-01-01

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

  8. Comparative architecture of octahedral protein cages. I. Indexed enclosing forms

    Science.gov (United States)

    Janner, A.

    2008-07-01

    The architecture of four protein cages (bacterio ferritin, human mitochondrial ferritin, sulfur oxygenase reductase and small heat-shock protein) are compared top-to-bottom, starting from polyhedra with vertices at cubic lattice points enclosing the cage down to indexed polyhedral forms of single monomers.

  9. Neighborhood Walkable Urban Form and C-Reactive Protein

    Science.gov (United States)

    Background: Walkable urban form predicts physical activity and lower body mass index, which lower C-reactive protein (CRP). However, urban form is also related to pollution, noise, social and health behavior, crowding, and other stressors, which may complement or contravene walka...

  10. In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

    Energy Technology Data Exchange (ETDEWEB)

    Ebrahimi, Seyyed Mahmoud, E-mail: smebrahimi@shirazu.ac.ir [Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box 14155-3651,Tehran (Iran, Islamic Republic of); Research Center of Virus and Vaccine, Baqiyatallah University of Medical Science, P.O.Box 14155-3651, Tehran (Iran, Islamic Republic of); Dabaghian, Mehran [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of); Tebianian, Majid [Department of Biotechnology, Razi Vaccine and Serum Research Institute (RVSRI), P.O. Box 31975/148, Karaj, Tehran (Iran, Islamic Republic of); Zabeh Jazi, Mohammad Hossein [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of)

    2012-08-15

    Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS-PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0-33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

  11. In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

    International Nuclear Information System (INIS)

    Ebrahimi, Seyyed Mahmoud; Dabaghian, Mehran; Tebianian, Majid; Zabeh Jazi, Mohammad Hossein

    2012-01-01

    Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS–PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0–33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

  12. Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

    2006-01-01

    Roč. 70, č. 2 (2006), s. 656-666 ISSN 0026-895X R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * muscarinic receptors * G-protein activation Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.469, year: 2006

  13. Four crystal forms of a Bence-Jones protein

    Energy Technology Data Exchange (ETDEWEB)

    Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander, E-mail: amcphers@uci.edu [Molecular Biology and Biochemistry, University of California, Irvine, 560 Steinhaus Hall, Irvine, CA 92697-3900 (United States)

    2005-01-01

    Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4{sub 3}2{sub 1}2 and P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3{sub 1}21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

  14. Four crystal forms of a Bence-Jones protein

    International Nuclear Information System (INIS)

    Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander

    2004-01-01

    Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4 3 2 1 2 and P2 1 2 1 2 1 , with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3 1 21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases

  15. Anti-influenza M2e antibody

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  16. Reactive species formed on proteins exposed to singlet oxygen

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2004-01-01

    Singlet oxygen ((1)O(2)) is believed to be generated in biological systems by a range of endogenous processes (e.g. enzymatic and chemical reactions) and exogenous stimuli (e.g. UV or visible light in the presence of a sensitiser). Kinetic data is consistent with proteins being a major target...... hydroperoxides, which can be reduced to the corresponding alcohols; other products arising from radical intermediates can also be generated, particularly in the presence of UV light and metal ions. With His side-chains, poorly characterised peroxides are also formed. Reaction with Met and Cys has been proposed...

  17. Protein imprinting and recognition via forming nanofilms on microbeads surfaces in aqueous media

    International Nuclear Information System (INIS)

    Lu Yan; Yan Changling; Wang Xuejing; Wang Gongke

    2009-01-01

    In this paler, we present a technique of forming nanofilms of poly-3-aminophenylboronic acid (pAPBA) on the surfaces of polystyrene (PS) microbeads for proteins (papain and trypsin) in aqueous. Papain was chosen as a model to study the feasibility of the technique and trypsin as an extension. Obtained core-shell microbeads were characterized using scanning electron microscopy (SEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and BET methods. The results show that pAPBA formed nanofilms (60-100 nm in thickness) on the surfaces of PS microbeads. The specific surface area of the papain-imprinted beads was about 180 m 2 g -1 and its pore size was 31 nm. These imprinted microbeads exhibit high recognition specificity and fast mass transfer kinetics. The specificity of these imprinted beads mainly originates from the spatial effect of imprinted sites. Because the protein-imprinted sites were located at, or close to, the surface, the imprinted beads have good site accessibility toward the template molecules. The facility of the imprinting protocol and the high recognition properties of imprinted microbeads make the approach an attractive solution to problems in the field of biotechnology.

  18. Structure of haze forming proteins in white wines: Vitis vinifera thaumatin-like proteins.

    Science.gov (United States)

    Marangon, Matteo; Van Sluyter, Steven C; Waters, Elizabeth J; Menz, Robert I

    2014-01-01

    Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.

  19. Structure of haze forming proteins in white wines: Vitis vinifera thaumatin-like proteins.

    Directory of Open Access Journals (Sweden)

    Matteo Marangon

    Full Text Available Grape thaumatin-like proteins (TLPs play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1, and having different unfolding temperatures (56 vs. 62°C, with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.

  20. Identification of the chemical forms of selenium in soy protein

    International Nuclear Information System (INIS)

    Rodibaugh, R.

    1989-01-01

    Soybeans (Glycine max. L. Merr., Century) were grown hydroponically and intrinsically radiolabeled with 75 Se, an isotope of selenium (Se). The isotope was provided as 75 Se-Na 2 SeO 3 during the reproductive stage of growth until onset of senescence. Harvested seeds were processed into defatted soy meal. Soluble proteins were extracted in 20mM Tris-HCl buffer and fractionated into 11S, 7S, and 2S protein fractions by isoelectric precipitation. The 11S and 7S globulins, containing the glycinin and conglycinin storage proteins respectively, constitute the majority of extractable soy proteins. These storage proteins are the predominant proteins in soy protein isolate frequently used in food for human consumption. Approximately 24% of the defatted meal was soluble protein and accounted for 65% of the radioactivity associated with the soybean meal. The 11S fraction contained approximately 31% of the extracted protein and 27% of the extracted radioactivity. The 7S fraction contained approximately 32% and 35% of the extractable protein and radioactivity, respectively. The 2S fraction, containing the sulfur (S)-rich trypsin inhibitors, accounted for 17% of the protein and 27% of the radioactivity extracted from the defatted soy meal. Purification of the storage proteins by gel filtration and affinity chromatography showed higher levels of radioactivity associated with glycinin than conglycinin. Purified 11S proteins contained 1.09 ng Se per mg protein while 7S proteins contained 0.36 ng Se per mg protein

  1. M2 to D2

    International Nuclear Information System (INIS)

    Mukhi, Sunil; Papageorgakis, Constantinos

    2008-01-01

    We examine the recently proposed ''3-algebra'' field theory for multiple M2-branes and show that when a scalar field valued in the 3-algebra develops a vacuum expectation value, the resulting Higgs mechanism has the novel effect of promoting topological (Chern-Simons) to dynamical (Yang-Mills) gauge fields. This leads to a precise derivation of the maximally supersymmetric Yang-Mills theory on multiple D2-branes and thereby provides a relationship between 3-algebras and Yang-Mills theories. We discuss the physical interpretation of this result.

  2. Underestimated Halogen Bonds Forming with Protein Backbone in Protein Data Bank.

    Science.gov (United States)

    Zhang, Qian; Xu, Zhijian; Shi, Jiye; Zhu, Weiliang

    2017-07-24

    Halogen bonds (XBs) are attracting increasing attention in biological systems. Protein Data Bank (PDB) archives experimentally determined XBs in biological macromolecules. However, no software for structure refinement in X-ray crystallography takes into account XBs, which might result in the weakening or even vanishing of experimentally determined XBs in PDB. In our previous study, we showed that side-chain XBs forming with protein side chains are underestimated in PDB on the basis of the phenomenon that the proportion of side-chain XBs to overall XBs decreases as structural resolution becomes lower and lower. However, whether the dominant backbone XBs forming with protein backbone are overlooked is still a mystery. Here, with the help of the ratio (R F ) of the observed XBs' frequency of occurrence to their frequency expected at random, we demonstrated that backbone XBs are largely overlooked in PDB, too. Furthermore, three cases were discovered possessing backbone XBs in high resolution structures while losing the XBs in low resolution structures. In the last two cases, even at 1.80 Å resolution, the backbone XBs were lost, manifesting the urgent need to consider XBs in the refinement process during X-ray crystallography study.

  3. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

    2014-01-01

    -ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

  4. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  5. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  6. Effect of cytosolic pH on inward currents reveals structural characteristics of the proton transport cycle in the influenza A protein M2 in cell-free membrane patches of Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Mattia L DiFrancesco

    Full Text Available Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. "Transinhibition", i.e., instead of an increase of unidirectional outward current with increasing cytosolic H(+ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H(+ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A "recycling step" implies that the protein undergoes conformational changes (assigned to Trp41 and Val27 during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H(+. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.

  7. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose......-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  8. Detection and partial identification of proteins in pearls formed in ...

    African Journals Online (AJOL)

    They were ground into a powder of >10,000 mesh followed by ultra-sonication and extraction in water for 4 h at room temperature. ... that one protein had significant sequence homology to a putative vitelline envelop receptor for lysine in the common marine mussel Mytilus edulis, and the other to the putative imaginal disc ...

  9. Amides are novel protein modifications formed by physiological sugars.

    Science.gov (United States)

    Glomb, M A; Pfahler, C

    2001-11-09

    The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.

  10. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The removal of the auto-inhibitory domain that negatively regulates the kinase activity in M. truncatula results in a constitutively-active form, inducing symbiotic responses in the absence of bacterial signals. In this study, we verified the functionality of a DMI3 variant and its ability to induce spontaneous nodules in M.

  11. Diarylthiophenes as inhibitors of the pore-forming protein perforin

    OpenAIRE

    Miller, Christian K.; Huttunen, Kristiina M.; Denny, William A.; Jaiswal, Jagdish K.; Ciccone, Annette; Browne, Kylie A.; Trapani, Joseph A.; Spicer, Julie A.

    2016-01-01

    Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure?activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a t...

  12. Structure of a second crystal form of Bence-Jones protein Loc: Strikingly different domain associations in two crystal forms of a single protein

    International Nuclear Information System (INIS)

    Schiffer, M.; Ainsworth, C.; Xu, Z.B.; Carperos, W.; Olsen, K.; Solomon, A.; Stevens, F.J.; Chang, C.H.

    1989-01-01

    The authors have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-angstrom resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding pocket. The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion. The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry

  13. Diarylthiophenes as inhibitors of the pore-forming protein perforin.

    Science.gov (United States)

    Miller, Christian K; Huttunen, Kristiina M; Denny, William A; Jaiswal, Jagdish K; Ciccone, Annette; Browne, Kylie A; Trapani, Joseph A; Spicer, Julie A

    2016-01-15

    Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Why do alpha-beta parallel proteins, like flavodoxins, form misfolded off-pathway intermediates?

    NARCIS (Netherlands)

    Nabuurs, S.M.

    2009-01-01

    The question: “Why do α-β parallel proteins, like flavodoxins, form misfolded off-pathway
    intermediates?" is the main subject of this thesis. A. vinelandii apoflavodoxin is chosen as protein
    of interest as it is a representative of α-β parallel proteins, which are widely prevalent in

  15. Engineering a pH responsive pore forming protein.

    Science.gov (United States)

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-02-08

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

  16. Engineering a pH responsive pore forming protein

    Science.gov (United States)

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-02-01

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

  17. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    Science.gov (United States)

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

  18. A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

    Science.gov (United States)

    Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

    2008-02-29

    Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

  19. Aggregated forms of bull seminal plasma proteins and their heparin-binding activity

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Ryšlavá, H.; Liberda, J.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 616-630 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : bull seminal plasma proteins * heparin-binding proteins * aggregated forms of proteins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  20. Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria

    NARCIS (Netherlands)

    Maagd, de R.A.; Bravo, A.; Berry, C.; Crickmore, N.; Schnepf, H.E.

    2003-01-01

    Gram-positive spore-forming entomopathogenic bacteria can utilize a large variety of protein toxins to help them invade, infect, and finally kill their hosts, through their action on the insect midgut. These toxins belong to a number of homology groups containing a diversity of protein structures

  1. The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ

    Directory of Open Access Journals (Sweden)

    Brann Jessica H

    2010-05-01

    Full Text Available Abstract Background The signal transduction cascade operational in the vomeronasal organ (VNO of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2, which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO. Results Homer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1 and receptor expression enhancing protein 1 (REEP1 were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay. Conclusions TRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

  2. Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

    2006-06-01

    To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

  3. A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.

    Directory of Open Access Journals (Sweden)

    Yun-Shiang Chang

    Full Text Available BACKGROUND: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus, is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. PRINCIPAL FINDINGS: In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. CONCLUSIONS: From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

  4. A proposal for M2-brane-anti-M2-brane action

    International Nuclear Information System (INIS)

    Garousi, Mohammad R.

    2010-01-01

    We propose a manifestly SO(8) invariant BF type Lagrangian for describing the dynamics of M2-brane-anti-M2-brane system in flat spacetime. When one of the scalars which satisfies a free-scalar equation takes a large expectation value, the M2-brane-anti-M2-brane action reduces to the tachyon DBI action of D2-brane-anti-D2-brane system in flat spacetime.

  5. Diversity and evolution of ABC proteins in mycorrhiza-forming fungi.

    Science.gov (United States)

    Kovalchuk, Andriy; Kohler, Annegret; Martin, Francis; Asiegbu, Fred O

    2015-12-28

    Transporter proteins are predicted to have an important role in the mycorrhizal symbiosis, due to the fact that this type of an interaction between plants and fungi requires a continuous nutrient and signalling exchange. ABC transporters are one of the large groups of transporter proteins found both in plants and in fungi. The crucial role of plant ABC transporters in the formation of the mycorrhizal symbiosis has been demonstrated recently. Some of the fungal ABC transporter-encoding genes are also induced during the mycorrhiza formation. However, no experimental evidences of the direct involvement of fungal ABC transporters in this process are available so far. To facilitate the identification of fungal ABC proteins with a potential role in the establishment of the mycorrhizal symbiosis, we have performed an inventory of the ABC protein-encoding genes in the genomes of 25 species of mycorrhiza-forming fungi. We have identified, manually annotated and curated more than 1300 gene models of putative ABC protein-encoding genes. Out of those, more than 1000 models are predicted to encode functional proteins, whereas about 300 models represent gene fragments or putative pseudogenes. We have also performed the phylogenetic analysis of the identified sequences. The sets of ABC proteins in the mycorrhiza-forming species were compared to the related saprotrophic or plant-pathogenic fungal species. Our results demonstrate the high diversity of ABC genes in the genomes of mycorrhiza-forming fungi. Via comparison of transcriptomics data from different species, we have identified candidate groups of ABC transporters that might have a role in the process of the mycorrhiza formation. Results of our inventory will facilitate the identification of fungal transporters with a role in the mycorrhiza formation. We also provide the first data on ABC protein-coding genes for the phylum Glomeromycota and for orders Pezizales, Atheliales, Cantharellales and Sebacinales, contributing to

  6. Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

    International Nuclear Information System (INIS)

    Raccosta, S.; Martorana, V.; Manno, M.; Blanco, M.; Roberts, C.J.

    2016-01-01

    The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic ph, lysozyme and α-chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway.

  7. Principles for designing proteins with cavities formed by curved β sheets

    Energy Technology Data Exchange (ETDEWEB)

    Marcos, Enrique; Basanta, Benjamin; Chidyausiku, Tamuka M.; Tang, Yuefeng; Oberdorfer, Gustav; Liu, Gaohua; Swapna, G. V. T.; Guan, Rongjin; Silva, Daniel-Adriano; Dou, Jiayi; Pereira, Jose Henrique; Xiao, Rong; Sankaran, Banumathi; Zwart, Peter H.; Montelione, Gaetano T.; Baker, David

    2017-01-12

    Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.

  8. Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

    Science.gov (United States)

    Caruso, Paola; Dunmore, Benjamin J; Schlosser, Kenny; Schoors, Sandra; Dos Santos, Claudia; Perez-Iratxeta, Carol; Lavoie, Jessie R; Zhang, Hui; Long, Lu; Flockton, Amanda R; Frid, Maria G; Upton, Paul D; D'Alessandro, Angelo; Hadinnapola, Charaka; Kiskin, Fedir N; Taha, Mohamad; Hurst, Liam A; Ormiston, Mark L; Hata, Akiko; Stenmark, Kurt R; Carmeliet, Peter; Stewart, Duncan J; Morrell, Nicholas W

    2017-12-19

    Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1 , and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Pulmonary vascular and

  9. Application of gamma irradiation on forming protein-based edible films

    International Nuclear Information System (INIS)

    Sabato, Susy Frey

    2000-01-01

    In the last decade considerable interest has been addressed to the development of protein-based edible films due to their application in the food industry, as a substitute to traditional plastic films. The use of soy and whey proteins to form those films has been investigated, using heat, chemical and enzymatic processes. Gamma irradiation was recently reported to form caseinate-based edible films, due to the increase of the cohesive strength of the proteins by the formation of cross-links. This work aimed to verify the role of the gamma irradiation in the process of forming edible films from soy protein isolate (SPI) alone and in complex mixtures, that is, mixed with whey protein isolate (WPI), with carbethoxymethyl cellulose (CMC) and with poly(vinyl)alcohol (PVA). Gamma irradiation treatment improved significantly the mechanical properties for all films. The mechanical behavior is strongly related to the formulation, showing synergy between the gamma irradiation and the CMC, mainly for SPI-based films. SPI-based films presented a trend to decrease the water vapor permeability values when irradiated. The CMC addition showed significant improvements on the permeability for films from SPI and from the mixture of SPI with WPI. (author)

  10. Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    Srsen Vlastimil

    2009-04-01

    Full Text Available Abstract Background Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. Results We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. Conclusion Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

  11. Evaluation of C-Reactive Protein Level in Patients with Pain Form of Temporomandibular Joint Dysfunction

    Directory of Open Access Journals (Sweden)

    Malgorzata Pihut

    2018-01-01

    Full Text Available Temporomandibular joint dysfunction is a functional disorder concerned with the abnormal functioning of the muscles of the stomatognathic system and temporomandibular joints involved in the dynamic movements of the jaw and surrounding structures. The aim of the study was to compare the level of C-reactive protein in patients with pain and painless forms of temporomandibular joint dysfunction. Materials and methods. The study group consisted of 72 patients who reported to the prosthetic treatment because of temporomandibular joint dysfunction. The study group included 36 patients with pain form of dysfunction, and the control group included 36 patients with painless form of disorder. Each patient underwent specialized examination of functional disorders in order to diagnose the type of dysfunction and was commissioned to carry out a study of the blood test concerned with evaluation of the C-reactive protein (CRP level in the same analytical laboratory. The results of the investigation were subjected to statistical analysis. The research obtained approval from the Ethics Committee of the Jagiellonian University (KBET/125/L/2013. Level of Evidence for primary research was established as type V. Results. The mean values of C-reactive protein levels in both groups were in the normal range and did not differ statistically significantly, which indicates the fact that the pain form of the temporomandibular joint disorders is not associated with inflammation of the soft tissues of the joint. Conclusion. Painful form of the temporomandibular joint dysfunctions is not connected with the inflammation of joints.

  12. Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

    Science.gov (United States)

    Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

    1999-01-08

    Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

  13. Activation of human natural killer cells by the soluble form of cellular prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  14. Evaluation of Enzymatically Modified Soy Protein Isolate Film Forming Solution and Film at Different Manufacturing Conditions.

    Science.gov (United States)

    Mohammad Zadeh, Elham; O'Keefe, Sean F; Kim, Young-Teck; Cho, Jin-Hun

    2018-04-01

    The effects of transglutaminase on soy protein isolate (SPI) film forming solution and films were investigated by rheological behavior and physicochemical properties based on different manufacturing conditions (enzyme treatments, enzyme incubation times, and protein denaturation temperatures). Enzymatic crosslinking reaction and changes in molecular weight distribution were confirmed by viscosity measurement and SDS-PAGE, respectively, compared to 2 controls: the nonenzyme treated and the deactivated enzyme treated. Films treated with both the enzyme and the deactivated enzyme showed significant increase in tensile strength (TS), percent elongation (%E), and initial contact angle of films compared to the nonenzyme control film due to the bulk stabilizers in the commercial enzyme. Water absorption property, protein solubility, Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopy revealed that enzyme treated SPI film matrix in the molecular structure level, resulted in the changes in physicochemical properties. Based on our observation, the enzymatic treatment at appropriate conditions is a practical and feasible way to control the physical properties of protein based biopolymeric film for many different scientific and industrial areas. Enzymes can make bridges selectively among different amino acids in the structure of protein matrix. Therefore, protein network is changed after enzyme treatment. The behavior of biopolymeric materials is dependent on the network structure to be suitable in different applications such as bioplastics applied in food and pharmaceutical products. In the current research, transglutaminase, as an enzyme, applied in soy protein matrix in different types of forms, activated and deactivated, and different preparation conditions to investigate its effects on different properties of the new bioplastic film. © 2018 Institute of Food Technologists®.

  15. Activation of human natural killer cells by the soluble form of cellular prion protein

    International Nuclear Information System (INIS)

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-01-01

    Cellular prion protein (PrP C ) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP C in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP C protein on human natural killer (NK) cells. Recombinant soluble PrP C protein was generated by fusion of human PrP C with the Fc portion of human IgG 1 (PrP C -Fc). PrP C -Fc binds to the surface of human NK cells, particularly to CD56 dim NK cells. PrP C -Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP C -Fc facilitated the IL-15-induced proliferation of NK cells. PrP C -Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP C -Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP C (PrP C -Fc) was generated by fusion of human PrP C with IgG1 Fc portion. • PrP C -Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP C -Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways

  16. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    International Nuclear Information System (INIS)

    Galat, Andrzej; Thai, Robert

    2014-01-01

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome

  17. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    Energy Technology Data Exchange (ETDEWEB)

    Galat, Andrzej, E-mail: galat@dsvidf.cea.fr; Thai, Robert

    2014-08-08

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

  18. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

    Full Text Available Proteins containing polyglutamine (polyQ regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD. To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

  19. The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

    Science.gov (United States)

    de Winter, J P; van der Weel, L; de Groot, J; Stone, S; Waisfisz, Q; Arwert, F; Scheper, R J; Kruyt, F A; Hoatlin, M E; Joenje, H

    2000-11-01

    Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

  20. Characteristics and potential role of M2 macrophages in COPD

    Directory of Open Access Journals (Sweden)

    He S

    2017-10-01

    Full Text Available Shengyang He, Lihua Xie, Junjuan Lu, Shenghua SunDepartment of Respiratory Medicine, The Third Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China Background: COPD is a multi-pathogenesis disease mainly caused by smoking. A further understanding of the mechanism of smoking-related COPD might contribute to preventions and treatments of this disease in the early stages. This study was designed to identify the characteristics of M2 macrophages in COPD for a better understanding about their potential role.Materials and methods: COPD models were built in the C57BL/6 mouse by cigarette smoke (CS exposure combined with intraperitoneal injection of cigarette smoke extract (CSE. The modeling efficiency was evaluated by lung function and hematoxylin and eosin (H&E staining. The number of different macrophage phenotypes was detected by immunohistochemical staining (IHS of CD206, CD86 and CD68 on the lung tissue paraffin section. The RAW264.7 cells were polarized toward the M2 phenotype by interleukin IL-4 and confirmed by a flow cytometer. The gene expression levels of TGF-βRII, Smad2, Smad3 and Smad7 in CSE-treated M2 macrophages were detected by real-time reverse transcription polymerase chain reaction (RT-PCR. The expression levels of TGF-β/Smad pathway-related makers (TGF-βRII, p-Smad2, p-Smad3, Smad7 and TGF-β in alveolar M2 macrophages were detected by two consecutive paraffin section IHS.Results: The COPD model is well established, which is confirmed by the lung function test and lung H&E staining. The whole number of macrophages and the ratio of M2/M1 phenotype are both increased (p<0.05. The level of CD206+ cells in IL-4-stimulated RAW264.7 cells is up to 93.4%, which is confirmed by a flow cytometer. The gene expression of TGF-βRII, Smad2, Smad3 and Smad7 are all enhanced (p<0.05 in CES-treated M2 macrophages, which is detected by RT-PCR. The protein levels of TGF-β/Smad pathway-related markers are

  1. Crystal Structure of the Oligomeric Form of Lassa Virus Matrix Protein Z.

    Science.gov (United States)

    Hastie, Kathryn M; Zandonatti, Michelle; Liu, Tong; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

    2016-05-01

    The arenavirus matrix protein Z is highly multifunctional and occurs in both monomeric and oligomeric forms. The crystal structure of a dodecamer of Z from Lassa virus, presented here, illustrates a ring-like structure with a highly basic center. Mutagenesis demonstrates that the dimeric interface within the dodecamer and a Lys-Trp-Lys triad at the center of the ring are important for oligomerization. This structure provides an additional template to explore the many functions of Z. The arenavirus Lassa virus causes hundreds of thousands of infections each year, many of which develop into fatal hemorrhagic fever. The arenavirus matrix protein Z is multifunctional, with at least four distinct roles. Z exists in both monomeric and oligomeric forms, each of which likely serves a specific function in the viral life cycle. Here we present the dodecameric form of Lassa virus Z and demonstrate that Z forms a "wreath" with a highly basic center. This structure and that of monomeric Z now provide a pair of critical templates by which the multiple roles of Z in the viral life cycle may be interpreted. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. M2 qualify laser beam propagation

    International Nuclear Information System (INIS)

    Abdelhalim, Bencheikh; Mohamed, Bouafia

    2010-01-01

    One of the most important properties of a laser resonator is the highly collimated or spatially coherent nature of the laser output beam. Laser beam diameter and quality factor M 2 are significant parameters in a wide range of laser applications. This is because the spatial beam quality determines how closely the beam can be focused or how well the beam propagates over long distances without significant dispersion. In the present paper we have used three different methods to qualify the spatial structure of a laser beam propagating in free space, the results are obtained and discussed, and we have found that the Wigner distribution function is a powerful tool which allows a global characterization of any kind of beam

  3. Piezo proteins are pore-forming subunits of mechanically activated channels.

    Science.gov (United States)

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-02-19

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  4. Comparing the photophysics of the two forms of the Orange Carotenoid Protein using 2D electronic spectroscopy

    Directory of Open Access Journals (Sweden)

    Mathies R.A.

    2013-03-01

    Full Text Available Broadband two-dimensional electronic spectroscopy is applied to investigate the photophysics of the photoactive orange carotenoid protein, which is involved in nonphotochemical quenching in cyanobacteria. Differences in dynamics between the light and dark forms arise from the different structure of the carotenoid in the protein pocket, with consequences for the biological role of the two forms.

  5. Expression and immunogenicity of M2e peptide of avian influenza virus H5N1 fused to ricin toxin B chain produced in duckweed plants

    Science.gov (United States)

    Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Shaloiko, Lyubov; Kozlov, Oleg; Vinokurov, Leonid; Rasskazova, Ekaterina; Murashev, Arkadii; Vainstein, Alexander; Dolgov, Sergey

    2018-02-01

    The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3’ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 µg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the dimeric form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of anti-M2e IgG was 1024. It was confirmed that oral immunization with RTB-M2e induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

  6. A primary microcephaly protein complex forms a ring around parental centrioles.

    Science.gov (United States)

    Sir, Joo-Hee; Barr, Alexis R; Nicholas, Adeline K; Carvalho, Ofelia P; Khurshid, Maryam; Sossick, Alex; Reichelt, Stefanie; D'Santos, Clive; Woods, C Geoffrey; Gergely, Fanni

    2011-10-09

    Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture and is caused by biallelic mutations in genes coding for a subset of centrosomal proteins. Although at least three of these proteins have been implicated in centrosome duplication, the nature of the centrosome dysfunction that underlies the neurodevelopmental defect in MCPH is unclear. Here we report a homozygous MCPH-causing mutation in human CEP63. CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor. Together, these two proteins are essential for maintaining normal centrosome numbers in cells. Using super-resolution microscopy, we found that CEP63 and CEP152 co-localize in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient cells derived from patients with MCPH. This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and that its impairment particularly affects human cerebral cortex growth.

  7. Diverse supramolecular structures formed by self‐assembling proteins of the B acillus subtilis spore coat

    Science.gov (United States)

    Jiang, Shuo; Wan, Qiang; Krajcikova, Daniela; Tang, Jilin; Tzokov, Svetomir B.; Barak, Imrich

    2015-01-01

    Summary Bacterial spores (endospores), such as those of the pathogens C lostridium difficile and B acillus anthracis, are uniquely stable cell forms, highly resistant to harsh environmental insults. B acillus subtilis is the best studied spore‐former and we have used it to address the question of how the spore coat is assembled from multiple components to form a robust, protective superstructure. B . subtilis coat proteins (CotY, CotE, CotV and CotW) expressed in E scherichia coli can arrange intracellularly into highly stable macro‐structures through processes of self‐assembly. Using electron microscopy, we demonstrate the capacity of these proteins to generate ordered one‐dimensional fibres, two‐dimensional sheets and three‐dimensional stacks. In one case (CotY), the high degree of order favours strong, cooperative intracellular disulfide cross‐linking. Assemblies of this kind could form exquisitely adapted building blocks for higher‐order assembly across all spore‐formers. These physically robust arrayed units could also have novel applications in nano‐biotechnology processes. PMID:25872412

  8. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

    DEFF Research Database (Denmark)

    Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

    2005-01-01

    MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B...... carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and Mre....... subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became...

  9. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    Science.gov (United States)

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  10. Killing machines: three pore-forming proteins of the immune system

    Science.gov (United States)

    McCormack, Ryan; de Armas, Lesley; Shiratsuchi, Motoaki

    2014-01-01

    The evolution of early multicellular eukaryotes 400–500 million years ago required a defensive strategy against microbial invasion. Pore-forming proteins containing the membrane-attack-complex-perforin (MACPF) domain were selected as the most efficient means to destroy bacteria or virally infected cells. The mechanism of pore formation by the MACPF domain is distinctive in that pore formation is purely physical and unspecific. The MACPF domain polymerizes, refolds, and inserts itself into bilayer membranes or bacterial outer cell walls. The displacement of surface lipid/carbohydrate molecules by the polymerizing MACPF domain creates clusters of large, water-filled holes that destabilize the barrier function and provide access for additional anti-bacterial or anti-viral effectors to sensitive sites that complete the destruction of the invader via enzymatic or chemical attack. The highly efficient mechanism of anti-microbial defense by a combined physical and chemical strategy using pore-forming MACPF-proteins has been retargeted during evolution of vertebrates and mammals for three purposes: (1) to kill extracellular bacteria C9/polyC9 evolved in conjunction with complement, (2) to kill virus infected and cancer cells perforin-1/polyperforin-1 CTL evolved targeted by NK and CTL, and (3) to kill intracellular bacteria transmembrane perforin-2/putative polyperforin-2 evolved targeted by phagocytic and nonphagocytic cells. Our laboratory has been involved in the discovery and description of each of the three pore-formers that will be reviewed here. PMID:24293008

  11. Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

    Science.gov (United States)

    Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

    2002-12-24

    KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

  12. Characterization of Protein and Peptide Binding to Nanogels Formed by Differently Charged Chitosan Derivatives

    Directory of Open Access Journals (Sweden)

    Anastasia Zubareva

    2013-07-01

    Full Text Available Chitosan (Chi is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC by a Ca2+ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.

  13. Penrose limits, pp waves, and deformed M2-branes

    International Nuclear Information System (INIS)

    Cvetic, M.; Lue, H.; Pope, C.N.

    2004-01-01

    Motivated by the recent discussions of the Penrose limit of AdS 5 xS 5 , we examine a more general class of supersymmetric pp-wave solutions of the type IIB theory, with a larger number of nonvanishing structures in the self-dual 5-form. One of the pp-wave solutions can be obtained as a Penrose limit of a D3-D3 intersection. In addition to 16 standard supersymmetries these backgrounds always allow for supernumerary supersymmetries. The latter are in one-to-one correspondence with the linearly realized world-sheet supersymmetries of the corresponding exactly solvable type IIB string action. The pp-waves provide new examples where supersymmetries will survive in a T-duality transformation on the x + coordinate. The T-dual solutions can be lifted to give supersymmetric deformed M2-branes in D=11. The deformed M2-brane is dual to a three-dimensional field theory whose renormalization group flow runs from the conformal fixed point in the infrared regime to a nonconformal theory as the energy increases. At a certain intermediate energy scale there is a phase transition associated with a naked singularity of the M2-brane. In the ultraviolet limit the theory is related by T duality to an exactly solvable massive type IIB string theory

  14. The proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel.

    Directory of Open Access Journals (Sweden)

    Michael Henkel

    2010-06-01

    Full Text Available PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization.Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2 derived from the IAV isolates A/Puerto Rico/8/34(H1N1 (IAV(PR8, from A/Brevig Mission/1/1918(H1N1 (IAV(SF2 or the H5N1 consensus sequence (IAV(BF2 were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns.The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.

  15. Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

    Science.gov (United States)

    Falik Zaccai, Tzipora C; Savitzki, David; Zivony-Elboum, Yifat; Vilboux, Thierry; Fitts, Eric C; Shoval, Yishay; Kalfon, Limor; Samra, Nadra; Keren, Zohar; Gross, Bella; Chasnyk, Natalia; Straussberg, Rachel; Mullikin, James C; Teer, Jamie K; Geiger, Dan; Kornitzer, Daniel; Bitterman-Deutsch, Ora; Samson, Abraham O; Wakamiya, Maki; Peterson, Johnny W; Kirtley, Michelle L; Pinchuk, Iryna V; Baze, Wallace B; Gahl, William A; Kleta, Robert; Anikster, Yair; Chopra, Ashok K

    2017-02-01

    Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A 2 -activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E 2 and cytosolic phospholipase A 2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E 2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E 2 and cytosolic phospholipase A 2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E 2 The non-functional phospholipase A 2 -activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email

  16. Electrophoretic protein profiles of mid-sized copepod Calanoides patagoniensis steadily fed bloom-forming diatoms

    Directory of Open Access Journals (Sweden)

    Victor M Aguilera

    2015-09-01

    Full Text Available Recent field and experimental evidence collected in the southern upwelling region off Concepción (36°5'S, 73°3'W showed an abrupt reduction (<72 h in the egg production rates (EPR of copepods when they were fed steadily and solely with the local bloom-forming diatom Thalassiosira rotula. Because diatoms were biochemically similar to dinoflagellate Prorocentrum minimum, a diet which supported higher reproductive outcomes, the fecundity reduction observed in copepod females fed with the diatom may have obeyed to post-ingestive processes, giving rise to resources reallocation. This hypothesis was tested by comparing feeding (clearance and ingestion rates, reproduction (EPR and hatching success and the structure of protein profiles (i.e., number and intensity of electrophoretic bands of copepods (adults and eggs incubated during 96 h with the two food conditions. The structure of protein profiles included molecular sizes that were calculated from the relative mobility of protein standards against the logarithm of their molecular sizes. After assessing the experimental conditions, feeding decreased over time for those females fed with T. rotula, while reproduction was higher in females fed with P. minimum. Electrophoretic profiles resulted similar mostly at a banding region of 100 to 89-kDa, while they showed partial differences around the region of 56-kDa band, especially in those females fed and eggs produced with T. rotula. Due to reproductive volume was impacted while larvae viability, a physiological processes with specific and high nutritional requirements, was independent on food type; post-ingestive processes, such as expression of stress-related proteins deviating resources to metabolic processes others than reproduction, are discussed under framework of nutritional-toxic mechanisms mediating copepod-diatoms relationships in productive upwelling areas.

  17. A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

    Directory of Open Access Journals (Sweden)

    M. Lauren Donnelly

    2017-01-01

    Full Text Available Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG, a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies.

  18. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

    Science.gov (United States)

    Johnson, Brant R.; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo

    2015-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  19. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.

    Science.gov (United States)

    Raghava, Smita; Giorda, Kristina M; Romano, Fabian B; Heuck, Alejandro P; Hebert, Daniel N

    2013-06-04

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.

  20. Destabilization of Oil-in-Water Emulsions Formed Using Highly Hydrolyzed Whey Proteins.

    Science.gov (United States)

    Agboola; Singh; Munro; Dalgleish; Singh

    1998-01-19

    Oil-in-water emulsions (4 wt % soy oil) were prepared with 0.5-5 wt % whey protein hydrolysate (WPH) (27% degree of hydrolysis), in a two-stage homogenizer using various first-stage pressures of 10.3, 20.6, and 34.3 MPa and a constant second-stage pressure of 3.4 MPa. Destabilization studies on the emulsions were carried out for up to 24 h, using both laser light scattering and confocal laser microscopy. It was found that emulsions formed with oiling off and coalescence at all homogenization pressures. Emulsions formed with 2, 3, and 4% WPH showed coalescence and creaming only, while slight flocculation but no creaming occurred in emulsions formed with 5% WPH. Furthermore, the apparent rate of coalescence increased with homogenization pressure but decreased with WPH concentration. In contrast, the surface concentration of WPH increased with the WPH concentration in the emulsions but decreased with homogenization pressure. Analysis of WPH by high-performance liquid chromatography showed an increase in the concentration of high molecular weight peptides at the droplet surface compared to the WPH solution. This was considered very important for the stability of these oil-in-water emulsions.

  1. Variation in Orthologous Shell-Forming Proteins Contribute to Molluscan Shell Diversity.

    Science.gov (United States)

    Jackson, Daniel J; Reim, Laurin; Randow, Clemens; Cerveau, Nicolas; Degnan, Bernard M; Fleck, Claudia

    2017-11-01

    Despite the evolutionary success and ancient heritage of the molluscan shell, little is known about the molecular details of its formation, evolutionary origins, or the interactions between the material properties of the shell and its organic constituents. In contrast to this dearth of information, a growing collection of molluscan shell-forming proteomes and transcriptomes suggest they are comprised of both deeply conserved, and lineage specific elements. Analyses of these sequence data sets have suggested that mechanisms such as exon shuffling, gene co-option, and gene family expansion facilitated the rapid evolution of shell-forming proteomes and supported the diversification of this phylum specific structure. In order to further investigate and test these ideas we have examined the molecular features and spatial expression patterns of two shell-forming genes (Lustrin and ML1A2) and coupled these observations with materials properties measurements of shells from a group of closely related gastropods (abalone). We find that the prominent "GS" domain of Lustrin, a domain believed to confer elastomeric properties to the shell, varies significantly in length between the species we investigated. Furthermore, the spatial expression patterns of Lustrin and ML1A2 also vary significantly between species, suggesting that both protein architecture, and the regulation of spatial gene expression patterns, are important drivers of molluscan shell evolution. Variation in these molecular features might relate to certain materials properties of the shells of these species. These insights reveal an important and underappreciated source of variation within shell-forming proteomes that must contribute to the diversity of molluscan shell phenotypes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. M2M Optimizations in Public Mobile Networks

    NARCIS (Netherlands)

    Norp, A.H.J.; Landais, B.

    2012-01-01

    Many M2M applications use public telecommunications networks to transfer data from M2M devices to an M2M server. These telecommunications networks will have to be adapted to cope with the traffic generated by the projected growth of M2M applications. In the near future, many more devices will be

  3. Characterization of the adenoassociated virus Rep protein complex formed on the viral origin of DNA replication

    International Nuclear Information System (INIS)

    Li Zengi; Brister, J. Rodney; Im, Dong-Soo; Muzyczka, Nicholas

    2003-01-01

    Interaction between the adenoassociated virus (AAV) replication proteins, Rep68 and 78, and the viral terminal repeats (TRs) is mediated by a DNA sequence termed the Rep-binding element (RBE). This element is necessary for Rep-mediated unwinding of duplex DNA substrates, directs Rep catalyzed cleavage of the AAV origin of DNA replication, and is required for viral transcription and proviral integration. Six discrete Rep complexes with the AAV TR substrates have been observed in vitro, and cross-linking studies suggest these complexes contain one to six molecules of Rep. However, the functional relationship between Rep oligomerization and biochemical activity is unclear. Here we have characterized Rep complexes that form on the AAV TR. Both Rep68 and Rep78 appear to form the same six complexes with the AAV TR, and ATP seems to stimulate formation of specific, higher order complexes. When the sizes of these Rep complexes were estimated on native polyacrylamide gels, the four slower migrating complexes were larger than predicted by an amount equivalent to one or two TRs. To resolve this discrepancy, the molar ratio of protein and DNA was calculated for the three largest complexes. Data from these experiments indicated that the larger complexes included multiple TRs in addition to multiple Rep molecules and that the Rep-to-TR ratio was approximately 2. The two largest complexes were also associated with increased Rep-mediated, origin cleavage activity. Finally, we characterized a second, Rep-mediated cleavage event that occurs adjacent to the normal nicking site, but on the opposite strand. This second site nicking event effectively results in double-stranded DNA cleavage at the normal nicking site

  4. How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

    Directory of Open Access Journals (Sweden)

    Angela Schwede

    2015-12-01

    Full Text Available Variations on the statement "the variant surface glycoprotein (VSG coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.

  5. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin.

    Science.gov (United States)

    Svitil, A L; Chadhain, S; Moore, J A; Kirchman, D L

    1997-02-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products.

  6. The L polymerase protein of parainfluenza virus 3 forms an oligomer and can interact with the heterologous Sendai virus L, P and C proteins

    International Nuclear Information System (INIS)

    Smallwood, Sherin; Moyer, Sue A.

    2004-01-01

    We recently showed that the L protein of Sendai virus is present as an oligomer in the active P-L polymerase complex [Smallwood et al., Virology 304 (2002) 235]. We now demonstrate using two different epitope tags that the L protein of a second respirovirus, human parainfluenza type 3 virus (PIV3), also forms an L-L complex. L oligomerization requires the coexpression of the differentially epitope tagged L proteins. By exploiting a series of C-terminal truncations the L-L binding site maps to the N-terminal half of L. There is some complex formation between the heterologous PIV3 and Sendai L and P proteins; however, the heterologous L protein does not function in transcription of either the PIV3 or Sendai template. The PIV3 C protein binds PIV3 L and inhibits RNA synthesis in vitro and in vivo. Significant homology exists between the C proteins of PIV3 and Sendai and complex formation occurs between the PIV3 and Sendai heterologous C and L proteins. In addition, the heterologous C proteins can inhibit transcription at ∼50% of the level of the homologous protein. These data suggest that while the C proteins may be functionally somewhat interchangeable, the L and P proteins are specific for each virus

  7. Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

    International Nuclear Information System (INIS)

    Montrose, Kristopher; Krissansen, Geoffrey W.

    2014-01-01

    Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC

  8. Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

    Energy Technology Data Exchange (ETDEWEB)

    Montrose, Kristopher; Krissansen, Geoffrey W., E-mail: gw.krissansen@auckland.ac.nz

    2014-10-31

    Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC.

  9. Cementum attachment protein manifestation is restricted to the mineralized tissue forming cells of the periodontium

    International Nuclear Information System (INIS)

    Bar-Kana, I.; Pitaru, S.; Savion, N.; Narayanan, A.S.

    1998-01-01

    The mechanisms that regulate cementogenesis are mainly unknown. A specific cementum attachment protein (CAP) has been recently partially characterized and found to be more efficient in supporting the attachment of alveolar bone cells (ABC) and periodontal ligament cells (PLC) than that of gingival fibroblasts (GF). The purpose of this study was to determine the capacity of human periodontal-derived cells to bind an express CAP and to relate these properties to their capacity to express alkaline phosphatase (AlP) and form mineralized tissue (MTF). ABC, PLC and GF were tested. Human stromal bone marrow cells (SBMC) and a cementoma-derived cell line (CC) served as controls. CAP binding was determined using 125 I-CAP. The amount of MTF was assessed by alizarin red staining and image analysis determination of the amount of red-stained material. AlP and CAP expression were examined by histochemistry and immuno-chemistry, respectively. The highest expression of CAP was observed in CC, followed by PLC and ABC in decreasing order, whereas SBMC and GF did not express CAP, SBMC manifested the highest CAP binding capacity followed by CC, ABC, PLC and GF. MTF and AlP manifestation were greatest in SBMC, followed by ABC, PLC and CC. Collectively, the results indicate that CAP binding and secretion are not linked and that CAP manifestation is restricted to periodontal derived cell lineages with the potential of forming mineralized tissues. (au)

  10. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

    Science.gov (United States)

    Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L P; Tattum, M Howard; Panico, Silvia; Clare, Daniel K; Collinge, John; Saibil, Helen R; Wadsworth, Jonathan D F

    2016-05-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP. © 2016 The Authors.

  11. M1 and M2 Monocytes in Rheumatoid Arthritis: A Contribution of Imbalance of M1/M2 Monocytes to Osteoclastogenesis

    Directory of Open Access Journals (Sweden)

    Shoichi Fukui

    2018-01-01

    Full Text Available ObjectivesWe investigated the relationships among M1 monocytes, M2 monocytes, osteoclast (OC differentiation ability, and clinical characteristics in patients with rheumatoid arthritis (RA.MethodsPeripheral blood mononuclear cells (PBMCs were isolated from RA patients and healthy donors, and we then investigated the number of M1 monocytes or M2 monocytes by fluorescence-activated cell sorting. We also obtained and cultured CD14-positive cells from PBMCs from RA patients and healthy donors to investigate OC differentiation in vitro.ResultsForty RA patients and 20 healthy donors were included. Twenty-two patients (55% were anticitrullinated protein antibody (ACPA positive. The median M1/M2 ratio was 0.59 (0.31–1.11, interquartile range. There were no significant differences between the RA patients and healthy donors. There was a positive correlation between the M1/M2 ratio and the differentiated OC number in vitro in RA patients (ρ = 0.81, p < 0.001. The ACPA-positive patients had significantly higher M1/M2 ratios in vivo (p = 0.028 and significantly greater numbers of OCs in vitro (p = 0.005 than the ACPA-negative patients. Multivariable regression analysis revealed that the M1/M2 ratio was the sole significant contribution factor to in vitro osteoclastogenesis. RA patients with M1/M2 ratios >1 (having relatively more M1 monocytes had higher C-reactive protein and erythrocyte sedimentation rates than RA patients with M1/M2 ratios ≤1. M1-dominant monocytes in vitro produced higher concentrations of interleukin-6 upon stimulation with lipopolysaccharide than M2 monocytes.ConclusionM1/M2 monocytes imbalance strongly contributes to osteoclastogenesis of RA patients. Our findings cast M1 and M2 monocyte subsets in a new light as a new target of treatments for RA to prevent progression of osteoclastic bone destruction.

  12. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  13. Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish

    OpenAIRE

    Stürmer, Claudia

    2011-01-01

    The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid...

  14. Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

    Science.gov (United States)

    Tayou, Junior; Wang, Qiang; Jang, Geeng-Fu; Pronin, Alexey N; Orlandi, Cesare; Martemyanov, Kirill A; Crabb, John W; Slepak, Vladlen Z

    2016-04-22

    RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Los Alamos National Laboratory; Velappan, Nileena [Los Alamos National Laboratory; Schmidt, Jurgen G [Los Alamos National Laboratory

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  16. Binding properties of a streptavidin layer formed on a biotinylated Langmuir–Schaefer film of unfolded protein

    Energy Technology Data Exchange (ETDEWEB)

    Furuno, Taiji, E-mail: t_furuno@a8.keio.jp

    2016-04-01

    A Langmuir monolayer of carbonic anhydrase (CA) unfolded at an air/water interface was transferred onto the hydrophobic surface of a silicon wafer by means of the Langmuir–Schaefer technique. The transferred CA film was biotinylated and was incubated in a streptavidin (SAv) solution to obtain a densely packed SAv layer by biotin–SAv linkage. Biotinylated proteins including ferritin, catalase, alcohol dehydrogenase, and carbonic anhydrase were incubated with the SAv layer and binding of these proteins was examined by atomic force microscopy. High-density binding of the biotinylated proteins was observed, whereas the amount of adsorbed non-biotinylated proteins was low or negligible. The SAv layer on the Langmuir–Schaefer film of unfolded protein could become a basic architecture for protein immobilization studies. - Highlights: • Langmuir–Schaefer film of carbonic anhydrase (LSF-CA) was biotinylated. • A densely packed streptavidin (SAv) layer was formed on the biotinylated LSF-CA. • Biotinylated proteins were bound to the SAv layer at high density. • Nonspecific adsorption of intact proteins to the SAv layer was weak. • Atomic force microscopy showed the binding of proteins at molecular resolution.

  17. Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes.

    OpenAIRE

    Hoch, D H; Romero-Mira, M; Ehrlich, B E; Finkelstein, A; DasGupta, B R; Simpson, L L

    1985-01-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These f...

  18. Conformational Plasticity of the Influenza A M2 Transmembrane Helix in Lipid Bilayers Under Varying pH, Drug Binding and Membrane Thickness

    Science.gov (United States)

    Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

    2010-01-01

    Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins. PMID:20883664

  19. Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C)

    DEFF Research Database (Denmark)

    Lukovic, Dunja; Plasencia, Inés; Taberner, Francisco J

    2006-01-01

    Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of ...

  20. Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

    NARCIS (Netherlands)

    Sluyter, Van S.C.; Warnock, N.I.; Schmidt, S.; Anderson, P.; Kan, van J.A.L.; Bacic, A.; Waters, E.J.

    2013-01-01

    White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the

  1. The promyelocytic leukemia gene product (PML) forms stable complexes with the retinoblastoma protein

    DEFF Research Database (Denmark)

    Alcalay, M; Tomassoni, L; Colombo, E

    1998-01-01

    PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor alpha (RAR alpha) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the ...

  2. Application of gamma irradiation on forming protein-based edible films; Aplicacao da irradiacao na formacao de filmes comestiveis proteicos

    Energy Technology Data Exchange (ETDEWEB)

    Sabato, Susy Frey

    2000-07-01

    In the last decade considerable interest has been addressed to the development of protein-based edible films due to their application in the food industry, as a substitute to traditional plastic films. The use of soy and whey proteins to form those films has been investigated, using heat, chemical and enzymatic processes. Gamma irradiation was recently reported to form caseinate-based edible films, due to the increase of the cohesive strength of the proteins by the formation of cross-links. This work aimed to verify the role of the gamma irradiation in the process of forming edible films from soy protein isolate (SPI) alone and in complex mixtures, that is, mixed with whey protein isolate (WPI), with carbethoxymethyl cellulose (CMC) and with poly(vinyl)alcohol (PVA). Gamma irradiation treatment improved significantly the mechanical properties for all films. The mechanical behavior is strongly related to the formulation, showing synergy between the gamma irradiation and the CMC, mainly for SPI-based films. SPI-based films presented a trend to decrease the water vapor permeability values when irradiated. The CMC addition showed significant improvements on the permeability for films from SPI and from the mixture of SPI with WPI. (author)

  3. Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

    2006-06-01

    To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated. Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

  4. Deletion of the M2-2 Gene from Avian Metapneumovirus Subgroup C (aMPV-C) Impairs Virus Replication and Immunogenicity in Turkeys

    Science.gov (United States)

    The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) virus contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in either viral RNA transcription or replication. To further characterize the f...

  5. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

    NARCIS (Netherlands)

    Sha, H.; Yang, L.; Liu, M.; Xia, S.; Liu, Y.; Liu, F.; Kersten, A.H.; Qi, L.

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in

  6. 14-3-3 Proteins and a 13-lipoxygenase form associations in a phosphorylation-dependent manner

    NARCIS (Netherlands)

    Holtman, W.L.; Roberts, M.R.; Wang, M.

    2000-01-01

    Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeijl and Wang (2000) FEBS Lett. 474, 48-52]. It was shown by both co-immunoprecipitations and surface-plasmon

  7. Physicochemical Characteristics of Protein-Enriched Restructured Beef Steaks with Phosphates, Transglutaminase, and Elasticised Package Forming

    Directory of Open Access Journals (Sweden)

    Sephora Baugreet

    2018-01-01

    Full Text Available Restructured beef steaks were formulated by adding protein-rich ingredients (pea protein isolate (PPI, rice protein (RP, and lentil flour (LF (at 4 and 8%, phosphate (0.2%, and two binding agents: 1% (TG and 0.15% (TS. The effects of their addition on the physicochemical properties of the beef steaks were investigated. Protein content of the RP8TG sample was significantly higher than that of the control in both the raw and cooked state. Raw LF4TS exhibited greater (P<0.01 a∗ values than the control; however, after the cooking process, L∗, a∗, and b∗ values were similar for all treatments. Textural assessment showed that elevating protein level increased (P<0.001 hardness, chewiness, cohesiveness, and gumminess in cooked restructured steaks. LF addition reduced all textural values assessed, indicating a strong plant protein effect on texture modification. The commercial binder produced a better bind in combination with protein ingredients. This facilitated the production of uniformed restructured beef steaks from low-value beef muscles with acceptable quality parameters using a novel process technology.

  8. 26 CFR 1.401(m)-2 - ACP test.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ACP test. 1.401(m)-2 Section 1.401(m)-2 Internal... TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(m)-2 ACP test. (a) Actual contribution percentage (ACP) test—(1) In general—(i) ACP test formula. A plan satisfies the ACP test for a plan year only...

  9. Towards Horizontal Architecture for Autonomic M2M Service Networks

    Directory of Open Access Journals (Sweden)

    Juhani Latvakoski

    2014-05-01

    Full Text Available Today, increasing number of industrial application cases rely on the Machine to Machine (M2M services exposed from physical devices. Such M2M services enable interaction of physical world with the core processes of company information systems. However, there are grand challenges related to complexity and “vertical silos” limiting the M2M market scale and interoperability. It is here expected that horizontal approach for the system architecture is required for solving these challenges. Therefore, a set of architectural principles and key enablers for the horizontal architecture have been specified in this work. A selected set of key enablers called as autonomic M2M manager, M2M service capabilities, M2M messaging system, M2M gateways towards energy constrained M2M asset devices and creation of trust to enable end-to-end security for M2M applications have been developed. The developed key enablers have been evaluated separately in different scenarios dealing with smart metering, car sharing and electric bike experiments. The evaluation results shows that the provided architectural principles, and developed key enablers establish a solid ground for future research and seem to enable communication between objects and applications, which are not initially been designed to communicate together. The aim as the next step in this research is to create a combined experimental system to evaluate the system interoperability and performance in a more detailed manner.

  10. Sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability.

    Science.gov (United States)

    Surya, Wahyu; Torres, Jaume

    2015-04-02

    Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.

  11. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    Science.gov (United States)

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

    Directory of Open Access Journals (Sweden)

    Aleksander F Sikorski

    2007-01-01

    Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

  13. Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms.

    Directory of Open Access Journals (Sweden)

    Becky J Proskocil

    Full Text Available BACKGROUND: Epidemiological studies link organophosphorus pesticide (OP exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. CONCLUSIONS/SIGNIFICANCE: These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

  14. Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Tomasz Łęga

    Full Text Available Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human-avian-swine-human M2e (M2eH-A-S-H peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system.

  15. Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa.

    Science.gov (United States)

    McElroy, Kerensa; Mouton, Laurence; Du Pasquier, Louis; Qi, Weihong; Ebert, Dieter

    2011-09-01

    Collagen-like proteins containing glycine-X-Y repeats have been identified in several pathogenic bacteria potentially involved in virulence. Recently, a collagen-like surface protein, Pcl1a, was identified in Pasteuria ramosa, a spore-forming parasite of Daphnia. Here we characterise 37 novel putative P. ramosa collagen-like protein genes (PCLs). PCR amplification and sequencing across 10 P. ramosa strains showed they were polymorphic, distinguishing genotypes matching known differences in Daphnia/P. ramosa interaction specificity. Thirty PCLs could be divided into four groups based on sequence similarity, conserved N- and C-terminal regions and G-X-Y repeat structure. Group 1, Group 2 and Group 3 PCLs formed triplets within the genome, with one member from each group represented in each triplet. Maximum-likelihood trees suggested that these groups arose through multiple instances of triplet duplication. For Group 1, 2, 3 and 4 PCLs, X was typically proline and Y typically threonine, consistent with other bacterial collagen-like proteins. The amino acid composition of Pcl2 closely resembled Pcl1a, with X typically being glutamic acid or aspartic acid and Y typically being lysine or glutamine. Pcl2 also showed sequence similarity to Pcl1a and contained a predicted signal peptide, cleavage site and transmembrane domain, suggesting that it is a surface protein. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Wu, Xiaodong; Qi, Xian; Liang, Mifang; Li, Chuan; Cardona, Carol J; Li, Dexin; Xing, Zheng

    2014-06-01

    Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNβ-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses. © FASEB.

  17. A Survey on M2M Service Networks

    Directory of Open Access Journals (Sweden)

    Juhani Latvakoski

    2014-11-01

    Full Text Available The number of industrial applications relying on the Machine to Machine (M2M services exposed from physical world has been increasing in recent years. Such M2M services enable communication of devices with the core processes of companies. However, there is a big challenge related to complexity and to application-specific M2M systems called “vertical silos”. This paper focuses on reviewing the technologies of M2M service networks and discussing approaches from the perspectives of M2M information and services, M2M communication and M2M security. Finally, a discussion on technologies and approaches potentially enabling future autonomic M2M service networks are provided. According to our conclusions, it is seen that clear definition of the architectural principles is needed to solve the “vertical silo” problem and then, proceeding towards enabling autonomic capabilities for solving complexity problem appears feasible. Several areas of future research have been identified, e.g., autonomic information based services, optimization of communications with limited capability devices, real-time messaging, creation of trust and end to end security, adaptability, reliability, performance, interoperability, and maintenance.

  18. A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation.

    Science.gov (United States)

    Kupfer, G; Naf, D; Garcia-Higuera, I; Wasik, J; Cheng, A; Yamashita, T; Tipping, A; Morgan, N; Mathew, C G; D'Andrea, A D

    1999-04-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A>C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wild-type FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway.

  19. Cul8/Rtt101 Forms a Variety of Protein Complexes That Regulate DNA Damage Response and Transcriptional Silencing*

    OpenAIRE

    Mimura, Satoru; Yamaguchi, Tsuyoshi; Ishii, Satoru; Noro, Emiko; Katsura, Tomoya; Obuse, Chikashi; Kamura, Takumi

    2010-01-01

    The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical anal...

  20. Potential Biomarker of Myofibrillar Protein Oxidation in Raw and Cooked Ham: 3-Nitrotyrosine Formed by Nitrosation.

    Science.gov (United States)

    Feng, Xianchao; Li, Chenyi; Ullah, Niamat; Hackman, Robert M; Chen, Lin; Zhou, Guanghong

    2015-12-30

    The stability of cured meat products is increased by the protection of its proteins from oxidation by sodium nitrite (NaNO2) during processing. This study investigated the effects of NaNO2 (0, 50, 100, 200, and 400 mg/kg) on the physiochemical and structural characteristics of myofibrillar protein (MP) in raw and cooked ham. The NaNO2 showed a dose-dependent antioxidant effect, by inhibiting carbonyl formation, dityrosine formation, and denaturation of MP, and a nitrosative effect, through the formation of 3-Nitrotyrosine (3-NT). The 3-NT content within MP of raw ham had distinct negative correlations with sulfhydryl content and surface hydrophobicity. The 3-NT content within MP of cooked ham had significantly negative correlations with carbonyl, sulfhydryl content and turbidity and had significantly positive correlations with disulfide content. These results indicated that 3-NT may be a potential marker for protein oxidation in raw and cooked cured meat products.

  1. Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

    Directory of Open Access Journals (Sweden)

    Rodrigo Diaz-Espinoza

    Full Text Available Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP, is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(- or SO(4(-2. The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc. The aggregates also exhibited PrP(Sc-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

  2. Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models.

    Science.gov (United States)

    Amano, Ken-Ichi; Yoshidome, Takashi; Iwaki, Mitsuhiro; Suzuki, Makoto; Kinoshita, Masahiro

    2010-07-28

    We report a new progress in elucidating the mechanism of the unidirectional movement of a linear-motor protein (e.g., myosin) along a filament (e.g., F-actin). The basic concept emphasized here is that a potential field is entropically formed for the protein on the filament immersed in solvent due to the effect of the translational displacement of solvent molecules. The entropic potential field is strongly dependent on geometric features of the protein and the filament, their overall shapes as well as details of the polyatomic structures. The features and the corresponding field are judiciously adjusted by the binding of adenosine triphosphate (ATP) to the protein, hydrolysis of ATP into adenosine diphosphate (ADP)+Pi, and release of Pi and ADP. As the first step, we propose the following physical picture: The potential field formed along the filament for the protein without the binding of ATP or ADP+Pi to it is largely different from that for the protein with the binding, and the directed movement is realized by repeated switches from one of the fields to the other. To illustrate the picture, we analyze the spatial distribution of the entropic potential between a large solute and a large body using the three-dimensional integral equation theory. The solute is modeled as a large hard sphere. Two model filaments are considered as the body: model 1 is a set of one-dimensionally connected large hard spheres and model 2 is a double helical structure formed by two sets of connected large hard spheres. The solute and the filament are immersed in small hard spheres forming the solvent. The major findings are as follows. The solute is strongly confined within a narrow space in contact with the filament. Within the space there are locations with sharply deep local potential minima along the filament, and the distance between two adjacent locations is equal to the diameter of the large spheres constituting the filament. The potential minima form a ringlike domain in model 1

  3. M2 polarization enhances silica nanoparticle uptake by macrophages

    Directory of Open Access Journals (Sweden)

    Jessica eHoppstädter

    2015-03-01

    Full Text Available While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth.We employed different models of M1 and M2 polarization: GM-CSF/LPS/IFN-gamma was used to generate primary human M1 cells and M-CSF/IL-10 to differentiate M2 monocyte-derived macrophages. PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-gamma and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø 26 and 41 nm and microparticles (Ø 1.75 µm was quantified. At the concentration used (50 µg/ml, silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human monocyte-derived macrophages compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages (TAM obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue.In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2

  4. Whirlin and PDZ domain-containing 7 (PDZD7) proteins are both required to form the quaternary protein complex associated with Usher syndrome type 2.

    Science.gov (United States)

    Chen, Qian; Zou, Junhuang; Shen, Zuolian; Zhang, Weiping; Yang, Jun

    2014-12-26

    Usher syndrome (USH) is the leading genetic cause of combined hearing and vision loss. Among the three USH clinical types, type 2 (USH2) occurs most commonly. USH2A, GPR98, and WHRN are three known causative genes of USH2, whereas PDZD7 is a modifier gene found in USH2 patients. The proteins encoded by these four USH genes have been proposed to form a multiprotein complex, the USH2 complex, due to interactions found among some of these proteins in vitro, their colocalization in vivo, and mutual dependence of some of these proteins for their normal in vivo localizations. However, evidence showing the formation of the USH2 complex is missing, and details on how this complex is formed remain elusive. Here, we systematically investigated interactions among the intracellular regions of the four USH proteins using colocalization, yeast two-hybrid, and pull-down assays. We show that multiple domains of the four USH proteins interact among one another. Importantly, both WHRN and PDZD7 are required for the complex formation with USH2A and GPR98. In this USH2 quaternary complex, WHRN prefers to bind to USH2A, whereas PDZD7 prefers to bind to GPR98. Interaction between WHRN and PDZD7 is the bridge between USH2A and GPR98. Additionally, the USH2 quaternary complex has a variable stoichiometry. These findings suggest that a non-obligate, short term, and dynamic USH2 quaternary protein complex may exist in vivo. Our work provides valuable insight into the physiological role of the USH2 complex in vivo and informs possible reconstruction of the USH2 complex for future therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein

    Science.gov (United States)

    Ko, Tzu-Ping; Liao, Yi-Ting; Hsu, Kai-Cheng

    2017-01-01

    DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms. PMID:29220372

  6. Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

    DEFF Research Database (Denmark)

    Selao, Tiago Toscano; Branca, Rui; Chae, Pil Seok

    2011-01-01

    of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially...

  7. Textural behavior of gels formed by rice starch and whey protein isolate: Concentration and crosshead velocities

    Directory of Open Access Journals (Sweden)

    Thiago Novaes Silva

    Full Text Available ABSTRACT Fabricated food gels involving the use of hydrocolloids are gaining polpularity as confectionery/convenience foods. Starch is commonly combined with a hydrocolloid (protein our polyssacharides, particularly in the food industry, since native starches generally do not have ideal properties for the preparation of food products. Therefore the texture studies of starch-protein mixtures could provide a new approach in producing starch-based food products, being thus acritical attribute that needs to be carefully adjusted to the consumer liking. This work investigated the texture and rheological properties of mixed gels of different concentrations of rice starch (15%, 17.5%, and 20% and whey protein isolate (0%, 3%, and 6% with different crosshead velocities (0.05, 5.0, and 10.0 mm/s using a Box-Behnken experimental design. The samples were submitted to uniaxial compression tests with 80% deformation in order to determinate the following rheological parameters: Young’s modulus, fracture stress, fracture deformation, recoverable energy, and apparent biaxial elongational viscosity. Gels with a higher rice starch concentration that were submitted to higher test velocities were more rigid and resistant, while the whey protein isolate concentration had little influence on these properties. The gels showed a higher recoverable energy when the crosshead velocity was higher, and the apparent biaxial elongational viscosity was also influenced by this factor. Therefore, mixed gels exhibit different properties depending on the rice starch concentration and crosshead velocity.

  8. Tubule-forming capacity of the movement proteins of alfalfa mosaic virus and brome mosaic virus

    NARCIS (Netherlands)

    Kasteel, D. T.; van der Wel, N. N.; Jansen, K. A.; Goldbach, R. W.; van Lent, J. W.

    1997-01-01

    The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a

  9. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Directory of Open Access Journals (Sweden)

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  10. TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

    Science.gov (United States)

    Smyth, Danielle J; Harcus, Yvonne; White, Madeleine P J; Gregory, William F; Nahler, Janina; Stephens, Ian; Toke-Bjolgerud, Edward; Hewitson, James P; Ivens, Alasdair; McSorley, Henry J; Maizels, Rick M

    2018-04-01

    We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-β. The Heligmosomoides polygyrus TGF-β Mimic (Hp-TGM) shares no homology to any TGF-β family member, however it binds the mammalian TGF-β receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-β activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation. Copyright © 2018. Published by Elsevier Ltd.

  11. A novel form of the membrane protein CD147 that contains an extra Ig-like domain and interacts homophilically

    Directory of Open Access Journals (Sweden)

    Brown Marion H

    2003-11-01

    Full Text Available Abstract Background CD147 is a broadly distributed integral membrane glycoprotein with two Ig-like domains implicated in a wide range of functions. It is associated at the cell surface with the monocarboxylate transporters MCT1 and 4 but interactions of the extracellular region have not been characterised. Results We report the characterisation of a form of CD147 with an additional membrane-distal Ig-like domain. In contrast to the two domain form, this three domain form of CD147 interacts homophilically. Surface plasmon resonance analysis using recombinant proteins showed that the interaction was of low affinity (KD ~ 40 μM and this is typical of many interactions between membrane proteins. cDNA for the 3 domain form are rare but have been identified in human and mouse retina. Conclusion The finding that the three domain form of CD147 has an extracellular ligand, that is it interacts homophilically, suggests this interaction may be important in aligning lactate transporters in the retina where lactate is an important metabolite.

  12. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    Science.gov (United States)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  13. The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

    International Nuclear Information System (INIS)

    Hong, H.; Patel, D.; Tamm, L.; van den Berg, B.

    2006-01-01

    Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM

  14. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    Science.gov (United States)

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  15. Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    C.Y. Nishikawa

    2012-02-01

    Full Text Available Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL. A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

  16. Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

    Energy Technology Data Exchange (ETDEWEB)

    Nishikawa, C.Y.; Araújo, L.M.; Kadowaki, M.A.S.; Monteiro, R.A.; Steffens, M.B.R.; Pedrosa, F.O.; Souza, E.M.; Chubatsu, L.S. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-01-27

    Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ{sup 54} factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH{sub 4}Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

  17. Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense.

    Science.gov (United States)

    Nishikawa, C Y; Araújo, L M; Kadowaki, M A S; Monteiro, R A; Steffens, M B R; Pedrosa, F O; Souza, E M; Chubatsu, L S

    2012-02-01

    Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

  18. Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

    International Nuclear Information System (INIS)

    Nishikawa, C.Y.; Araújo, L.M.; Kadowaki, M.A.S.; Monteiro, R.A.; Steffens, M.B.R.; Pedrosa, F.O.; Souza, E.M.; Chubatsu, L.S.

    2012-01-01

    Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ 54 factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH 4 Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription

  19. Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

    Czech Academy of Sciences Publication Activity Database

    Ostatná, Veronika; Černocká, Hana; Kurzatkowska, K.; Paleček, Emil

    2012-01-01

    Roč. 735, JUL (2012), s. 31-36 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038 Institutional research plan: CEZ:AV0Z50040702 Keywords : protein denaturation * carbon electrodes * edge plane pyrolytic graphite Subject RIV: BO - Biophysics Impact factor: 4.387, year: 2012

  20. Deletion of the M2-2 gene from avian metapneumovirus subgroup C impairs virus replication and immunogenicity in Turkeys.

    Science.gov (United States)

    Yu, Qingzhong; Estevez, Carlos N; Roth, Jason P; Hu, Haixia; Zsak, Laszlo

    2011-06-01

    The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.

  1. Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

    Directory of Open Access Journals (Sweden)

    Shweta Joshi

    Full Text Available Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

  2. TNF Counterbalances the Emergence of M2 Tumor Macrophages

    Directory of Open Access Journals (Sweden)

    Franz Kratochvill

    2015-09-01

    Full Text Available Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

  3. Theoretical Assessment of 178m2Hf De-Excitation

    Energy Technology Data Exchange (ETDEWEB)

    Hartouni, E P; Chen, M; Descalle, M A; Escher, J E; Loshak, A; Navratil, P; Ormand, W E; Pruet, J; Thompson, I J; Wang, T F

    2008-10-06

    This document contains a comprehensive literature review in support of the theoretical assessment of the {sup 178m2}Hf de-excitation, as well as a rigorous description of controlled energy release from an isomeric nuclear state.

  4. A distributed approach for secure M2M communications

    OpenAIRE

    BEN SAIED , Yosra; OLIVEREAU , Alexis; LAURENT , Maryline

    2012-01-01

    International audience; A key establishment solution for heterogeneous Machine to Machine (M2M) communications is proposed. Decentralization in M2M environment leads to situations where highly resource-constrained nodes have to establish end-to-end secured contexts with powerful remote servers, which would normally be impossible because of the technological gap between these classes of devices. This paper proposes a novel collaborative session key exchange method, wherein a highly resource-co...

  5. Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109.

    Science.gov (United States)

    Abdali, Narges; Younas, Farhan; Mafakheri, Samaneh; Pothula, Karunakar R; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

    2018-05-09

    Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene

  6. The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

    Science.gov (United States)

    Bourdet, Isabelle; Preat, Thomas; Goguel, Valérie

    2015-01-21

    The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes. Copyright © 2015 the authors 0270-6474/15/351043-09$15.00/0.

  7. LEU WWR-M2 fuel assemblies burnable test

    International Nuclear Information System (INIS)

    Kirsanov, G.A.; Konoplev, K.A.; Pikulik, R.G.; Sajkov, Yu. P.; Tchmshkyan, D.V.; Tedoradze, L.V.; Zakharov, A.S.

    2000-01-01

    The results of in-pile irradiation tests of LEU WWR-M2 fuel assemblies with reduced enrichment of fuel are submitted in the report. The tests are made according to the Russian Program on Reduced Enrichment for Research and Test Reactors (RERTR). United States Department of Energy and the Ministry of Atomic Energy of Russian Federation jointly fund this Program. The irradiation tests of 5 WWR-M2 experimental assemblies are carried out at WWR-M reactor of the Petersburg Nuclear Physics Institute (PNPI). The information on assembly design and technique of irradiation tests is presented. In the irradiation tests the integrity of fuel assemblies is periodically measured. The report presents the data for the integrity maintained during the burnup of 5 fuel assemblies up to 45%. These results demonstrate the high reliability of the experimental fuel assemblies within the guaranteed burnup limits specified by the manufacturer. The tests are still in progress; it is planned to test and analyze the change in integrity for burnup of up to 70% - 75% or more. LEU WWR-M2 fuel assemblies are to be offered for export by their Novosibirsk manufacturer. Currently, HEU WWR-M2 fuel assemblies are used in Hungary, Ukraine and Vietnam. LEU WWR-M2 fuel assemblies were designed as a possible replacement for the HEU WWR-M2 fuel assemblies in those countries, but their use can be extended to other research reactors. (author)

  8. An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro*

    OpenAIRE

    Mason, Aaron C.; Haring, Stuart J.; Pryor, John M.; Staloch, Cathy A.; Gan, Tze Fei; Wold, Marc S.

    2009-01-01

    Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The “canonical” RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting “alternative” RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the c...

  9. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-04-19

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

  10. Effect of physical form and protein source of starter feed on growth and development of dairy calves

    Directory of Open Access Journals (Sweden)

    E. Yavuz

    2015-06-01

    Full Text Available Abstract. The objective of this paper was to review the literature on effects of different methods of processing of grain fraction of the starter feeds for young dairy calves, as well as providing another part of starter which is a source of protein, minerals and vitamins in different physical forms. The second aim was to discus the impact of the main protein sources for starter feeds on performance of preweaning and posteweaning dairy calves. The main criteria for assessment of physical form effect and sources of protein in the starter feeds were intake of dry feeds, daily live weight gain and frame size growth of calves, morphological and functional development of forestomachs, digestibility of feeds and health status of young calves. Data show big variations and lack of consistency of experimental results. Good results were achieved when calves were fed whole, ground, dry-rolled, pelleted and steam-flaked grains. It seemed that fineness of grinding and quantities of fine fraction were important for starter intake. Steam-flaking and grinding improved digestibility to the same extent, but whole grain stimulated chewing and improved rumen environment for bacteria growth. Soybean meal was the most palatable and ensured best performance of calves. Evidently, it is possible to replace soybean meal with rape seed, canola type meal, dry distillers grain with solubles (DDGS and other protein sources which contain more fiber and are less digestible. However, it is difficult to appraise how much and at what conditions is it possible to replace completely or a maximum possible portion of soybean meal. Additional studies are needed to clarify interactions between physical form of starters, rumen fermentation environments and age of calves. Information for composition of diets is needed allowing inclusion of maximum amounts of canola meal, DDGS and sunflower meal, which are produced locally and are cheaper than soybean meal, without decreasing live weight

  11. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    Directory of Open Access Journals (Sweden)

    Vuento Matti

    2006-12-01

    Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  12. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  13. Protein/lipid coaggregates are formed during α-synuclein-induced disruption of lipid bilayers

    DEFF Research Database (Denmark)

    van Maarschalkerweerd, Andreas; Vetri, Valeria; Langkilde, Annette Eva

    2014-01-01

    Amyloid formation is associated with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including the molec......Amyloid formation is associated with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including...... the molecular mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, we present experimental data based on hybrid analysis of two-photon-microscopy, solution...... small-angle X-ray scattering and circular dichroism data. Data show in real time changes in liposome morphology and stability upon protein addition and reveal that membrane disruption mediated by amyloidogenic αSN is associated with dehydration of anionic lipid membranes and stimulation of protein...

  14. Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

    Energy Technology Data Exchange (ETDEWEB)

    Cady, Sarah D.; Hong Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu

    2009-09-15

    The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the {sup 1}H rotating-frame spin-lattice relaxation (T{sub 1{rho}}) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by {sup 2}H, {sup 15}N and {sup 13}C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent {sup 1}H T{sub 1{rho}} relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 {+-} 0.9 {mu}s for the apo peptide to 0.89 {+-} 0.41 {mu}s for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln (T{sub 1{rho}}{sup -1}) indicates that the activation energy of motion increased from 14.0 {+-} 4.0 kJ/mol for the apo peptide to 23.3 {+-} 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

  15. Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

    Science.gov (United States)

    Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

    1998-01-01

    In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

  16. Determination of protein-bound-radioiodine level as an index for determining the form of thyrotoxicosis

    International Nuclear Information System (INIS)

    Peshev, N.; Mladenov, B.; Guginska, E.

    1977-01-01

    The diagnostic capabilities of the test in hyperthyroidism and its possibilities in determining the form and stage of the disease were studied on a rather ample case material of 243 patients. The major diagnostic value of the method in differentiating between euthyroid function and hyperthyroidism is pointed out. The great sensitivity of the test will allow detection of even the slightest disturbances of iodine metabolism. (author)

  17. A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein.

    Science.gov (United States)

    Crimmins, Dan L; Kao, Jeffrey L-F

    2008-07-01

    The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general. A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays.

  18. Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

    Science.gov (United States)

    Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

    2015-03-01

    In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

  19. Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli.

    Directory of Open Access Journals (Sweden)

    Sang Beum Lee

    Full Text Available Myostatin (MSTN is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42-175 (MBP-Pro42-175 exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42-115 (MBP-Pro42-115 or 42-98 (MBP-Pro42-98 also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42-175 is sufficient to maintain the full MSTN-inhibitory capacity.

  20. Molecular Cloning, Characterization, and Expression of Cuc m 2, a Major Allergen in Cucumis melo

    Directory of Open Access Journals (Sweden)

    Mojtaba Sankian

    2013-05-01

    Full Text Available Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins.

  1. An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

    Science.gov (United States)

    Yuan, Meng; McNae, Iain W; Chen, Yiyuan; Blackburn, Elizabeth A; Wear, Martin A; Michels, Paul A M; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

    2018-05-10

    We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t 1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate. ©2018 The Author(s).

  2. Comparison of radiation-induced DNA-protein cross-links formed in oxic, hypoxic, and glutathione depleted cells

    International Nuclear Information System (INIS)

    Xue, L.; Friedman, L.R.; Chiu, S.; Ramakrishnan, N.; Oleinick, N.L.

    1987-01-01

    Treatment of cells with L-buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH). Subsequent metabolism depletes the cells of GSH. GSH-depletion sensitizes both oxic and hypoxic cells to the lethal effects of ionizing radiation. DNA-protein cross-links (DPC) are formed preferentially between DNA sequences active in transcription and a subset of proteins of the nuclear matrix. Thus, DPC may be an indicator lesion of damage in sensitive regions of the genome. The interrelationships between GSH level, oxic vs. hypoxic status, and the yield of DPC have been studied in terms of number of lesions and repair rate in Chinese hamster V79 and in human lung carcinoma A549 cells. The data suggest that elevated background levels of DPC are indicative of a reduced repair capacity, and greater radiation-induced yields of DPC in hypoxia may also be indicative of a compromised repair mechanism

  3. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  4. Critical illness induces alternative activation of M2 macrophages in adipose tissue.

    Science.gov (United States)

    Langouche, Lies; Marques, Mirna B; Ingels, Catherine; Gunst, Jan; Derde, Sarah; Vander Perre, Sarah; D'Hoore, André; Van den Berghe, Greet

    2011-01-01

    We recently reported macrophage accumulation in adipose tissue of critically ill patients. Classically activated macrophage accumulation in adipose tissue is a known feature of obesity, where it is linked with increasing insulin resistance. However, the characteristics of adipose tissue macrophage accumulation in critical illness remain unknown. We studied macrophage markers with immunostaining and gene expression in visceral and subcutaneous adipose tissue from healthy control subjects (n = 20) and non-surviving prolonged critically ill patients (n = 61). For comparison, also subcutaneous in vivo adipose tissue biopsies were studied from 15 prolonged critically ill patients. Subcutaneous and visceral adipose tissue biopsies from non-surviving prolonged critically ill patients displayed a large increase in macrophage staining. This staining corresponded with elevated gene expression of "alternatively activated" M2 macrophage markers arginase-1, IL-10 and CD163 and low levels of the "classically activated" M1 macrophage markers tumor necrosis factor (TNF)-α and inducible nitric-oxide synthase (iNOS). Immunostaining for CD163 confirmed positive M2 macrophage staining in both visceral and subcutaneous adipose tissue biopsies from critically ill patients. Surprisingly, circulating levels and tissue gene expression of the alternative M2 activators IL-4 and IL-13 were low and not different from controls. In contrast, adipose tissue protein levels of peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor required for M2 differentiation and acting downstream of IL-4, was markedly elevated in illness. In subcutaneous abdominal adipose tissue biopsies from surviving critically ill patients, we could confirm positive macrophage staining with CD68 and CD163. We also could confirm elevated arginase-1 gene expression and elevated PPARγ protein levels. Unlike obesity, critical illness evokes adipose tissue accumulation of alternatively activated M2

  5. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders.

    Science.gov (United States)

    Andreeva, Liudmila; Hiller, Björn; Kostrewa, Dirk; Lässig, Charlotte; de Oliveira Mann, Carina C; Jan Drexler, David; Maiser, Andreas; Gaidt, Moritz; Leonhardt, Heinrich; Hornung, Veit; Hopfner, Karl-Peter

    2017-09-21

    Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA and pathogen genomes, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.

  6. Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Zhili Liang

    2016-07-01

    Full Text Available Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs in foods. Peptide-enriched drinks (PEDs are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr. In this study we determined free-form pyrraline (Free-Pyr and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH, soy protein hydrolysate (SPH and collagen protein hydrolysate (CPH. A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE. The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

  7. Main: 1M2Q [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1M2Q トウモロコシ Corn Zea mays L. Casein Kinase Ii, Alpha Chain Name=Ack2; Zea Mays Mole...cule: Casein Kinase Ii, Alpha Chain; Chain: A; Fragment: Catlytic Subunit; Synonym: Ckii; Engineered: Yes Tr...ansferase 2.7.1.37 (Casein Kinase Ii, Alpha Chain) E.De Moliner, S.Sarno, S.Moro, G.Zagotto, G.Zanotti, L.A....=2-326.|PDB; 1M2Q; X-ray; A=2-328.|PDB; 1M2R; X-ray; A=2-328.|PDB; 1OM1; X-ray; A=1-332.|Mai

  8. Polarized M2 macrophages in dogs with visceral leishmaniasis.

    Science.gov (United States)

    Moreira, Pamela Rodrigues Reina; Fernando, Filipe Santos; Montassier, Hélio José; André, Marcos Rogério; de Oliveira Vasconcelos, Rosemeri

    2016-08-15

    The objective of the present study was to analyze the skin (nasal surface and ear regions), lymph nodes (popliteal and pre-scapular), spleen and liver of dogs with visceral leishmaniasis (VL), in order to investigate the relationship between the parasite load measured as DNA copy number of Alpha gene of DNA polymerase of Leishmania infantum by quantitative PCR and the number of M2 macrophages by immunohistochemistry. A set of 29 naturally infected dogs from an endemic area for VL were sampled and another set of six dogs negative for VL and from a non-endemic area were analyzed as the control group (C). The spleen presented the highest number of Leishmania DNA copies, with significant differences between the groups G1 and G2 (with and without skin lesions, respectively). The M2 phenotype immunostaining predominated among the macrophages in granulomas and inflammatory infiltrates of samples from the skin, lymph nodes and spleens examined. The presence of M2 macrophages in dogs from infected group differed significantly from the control group, in all organs analyzed, excepted liver. The highest proportion of M2 macrophages coincided with the highest parasitism loads found in more susceptible organs of VL dogs, even in the skin, considered a more resistant organ, while the liver showed low parasitism load and low immunostaining for M2 macrophages with no significant differences between infected and negative groups. It was concluded that the predominance of M2 phenotype in VL dogs favored the multiplication of Leishmania infantum in organs of dogs that are more susceptible to Leishmania infection, as skin, lymph nodes and spleen. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Carbides crystalline structure of AISI M2 high-speed steel

    International Nuclear Information System (INIS)

    Serna, M.M.; Galego, E.; Rossi, J.L.

    2005-01-01

    The aim of this study was to identify the crystallographic structure of the extracted carbides of AISI M2 steel spray formed The structure determination of these carbides. The structure determination of these carbides is a very hard work. Since these structures were formed by atom migration it is not possible to reproduce them by a controlled process with a determined chemical composition. The solution of this problem is to obtain the carbide by chemical extraction from the steel. (Author)

  10. Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

    Science.gov (United States)

    Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

    2002-01-01

    The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

  11. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

    Science.gov (United States)

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-01-01

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

  12. Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion.

    Science.gov (United States)

    Chen, Yu; Deng, Xiaoling; Deng, Junfang; Zhou, Jiehua; Ren, Yuping; Liu, Shengxuan; Prusak, Deborah J; Wood, Thomas G; Bao, Xiaoyong

    2016-12-01

    Human metapneumovirus (hMPV) is a major cause of lower respiratory infection in young children. Repeated infections occur throughout life, but its immune evasion mechanisms are largely unknown. We recently found that hMPV M2-2 protein elicits immune evasion by targeting mitochondrial antiviral-signaling protein (MAVS), an antiviral signaling molecule. However, the molecular mechanisms underlying such inhibition are not known. Our mutagenesis studies revealed that PDZ-binding motifs, 29-DEMI-32 and 39-KEALSDGI-46, located in an immune inhibitory region of M2-2, are responsible for M2-2-mediated immune evasion. We also found both motifs prevent TRAF5 and TRAF6, the MAVS downstream adaptors, to be recruited to MAVS, while the motif 39-KEALSDGI-46 also blocks TRAF3 migrating to MAVS. In parallel, these TRAFs are important in activating transcription factors NF-kB and/or IRF-3 by hMPV. Our findings collectively demonstrate that M2-2 uses its PDZ motifs to launch the hMPV immune evasion through blocking the interaction of MAVS and its downstream TRAFs. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. A nonperturbative test of M2-brane theory

    International Nuclear Information System (INIS)

    Hosomichi, Kazuo; Lee, Ki-Myeong; Lee, Sungjay; Yi, Piljin; Lee, Sangmin; Park, Jaemo

    2008-01-01

    We discuss non-perturbative effects in the ABJM model due to monopole instantons. We begin by constructing the instanton solutions in the U(2) x U(2) model, explicitly, and computing the Euclidean action. The Wick-rotated Lagrangian is complex and its BPS monopole instantons are found to be a delicate version of the usual 't Hooft-Polyakov monopole solutions. They are generically 1/3 BPS but become 1/2 BPS at special locus in the moduli space of two M2-branes, yet each instanton carries eight fermionic zero modes, regardless of the vacuum choice. The low energy effective action induced by monopole instantons are quartic order in derivatives. The resulting vertices are nonperturbative in 1/k, as expected, but are rational functions of the vacuum moduli. We also analyze the system of two M2-branes in the supergravity framework and compute the higher order interactions via 11-dimensional supergraviton exchange. The comparison of the two shows that the instanton vertices are precisely reproduced by this M2-brane picture, supporting the proposal that the ABJM model describes multiple M2-branes.

  14. Dimeric Complexes of Tryptophan with M2+ Metal Ions

    NARCIS (Netherlands)

    Dunbar, R. C.; Steill, J. D.; Polfer, N. C.; Oomens, J.

    2009-01-01

    IRMPD spectroscopy using the FELIX free electron laser and a Fourier transform ICR mass spectrometer was used to characterize the structures of electrosprayed dimer complexes M(2+)Trp(2) of tryptophan with a series of eight doubly charged metal ions, including alkaline earths Ca, Sr, and Ba, and

  15. Multiple M2-branes and the embedding tensor

    NARCIS (Netherlands)

    Bergshoeff, Eric A.; de Roo, Mees; Hohm, Olaf

    2008-01-01

    We show that the Bagger-Lambert theory of multiple M2-branes fits into the general construction of maximally supersymmetric gauge theories using the embedding tensor technique. We apply the embedding tensor technique in order to systematically obtain the consistent gaugings of N = 8 superconformal

  16. An N=1 superfield action for M2 branes

    International Nuclear Information System (INIS)

    Mauri, Andrea; Petkou, Anastasios C.

    2008-01-01

    We present an octonionic N=1 superfield action that reproduces in components the action of Bagger and Lambert for M2 branes. By giving an expectation value to one of the scalars we obtain the maximally supersymmetric superfield action for D2 branes

  17. Toxicity and oxidative stress of different forms of organic selenium (Se) and dietary protein in mallard (Anas platyrhynchos) ducklings

    Science.gov (United States)

    Hoffman, D.; Heinz, G.; Eisemann, J.; Pendleton, G.

    1994-01-01

    High concentrations of Se have been found in aquatic food chains associated with irrigation drainwater and toxicity to fish and wildlife. Earlier studies have compared toxicities of Se as selenite and as seleno-DL-methionine (DL) in mallards. This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W). Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms. After 2 weeks blood and liver samples were collected for biochemical assays and Se analysis. All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities. Se as L was the most toxic, resulting in high mortality (64%) and impaired growth (>50%) and the greatest increase in ratio of oxidized to reduced glutathione with 30 ppm in the diet. Se as Y accumulated the least in liver. In a subsequent experiment with 30% dietary protein Se as L was less toxic.

  18. Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

    Directory of Open Access Journals (Sweden)

    Youn-Bok Lee

    2013-12-01

    Full Text Available The GGGGCC (G4C2 intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  19. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    International Nuclear Information System (INIS)

    Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Urzhumtsev, Alexandre; Gualerzi, Claudio O.; Klaholz, Bruno P.

    2013-01-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue

  20. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    Energy Technology Data Exchange (ETDEWEB)

    Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  1. A Corticocortical Circuit Directly Links Retrosplenial Cortex to M2 in the Mouse

    Science.gov (United States)

    Radulovic, Jelena

    2016-01-01

    Retrosplenial cortex (RSC) is a dorsomedial parietal area involved in a range of cognitive functions, including episodic memory, navigation, and spatial memory. Anatomically, the RSC receives inputs from dorsal hippocampal networks and in turn projects to medial neocortical areas. A particularly prominent projection extends rostrally to the posterior secondary motor cortex (M2), suggesting a functional corticocortical link from the RSC to M2 and thus a bridge between hippocampal and neocortical networks involved in mnemonic and sensorimotor aspects of navigation. We investigated the cellular connectivity in this RSC→M2 projection in the mouse using optogenetic photostimulation, retrograde labeling, and electrophysiology. Axons from RSC formed monosynaptic excitatory connections onto M2 pyramidal neurons across layers and projection classes, including corticocortical/intratelencephalic neurons (reciprocally and callosally projecting) in layers 2–6, pyramidal tract neurons (corticocollicular, corticopontine) in layer 5B, and, to a lesser extent, corticothalamic neurons in layer 6. In addition to these direct connections, disynaptic connections were made via posterior parietal cortex (RSC→PPC→M2) and anteromedial thalamus (RSC→AM→M2). In the reverse direction, axons from M2 monosynaptically excited M2-projecting corticocortical neurons in the RSC, especially in the superficial layers of the dysgranular region. These findings establish an excitatory RSC→M2 corticocortical circuit that engages diverse types of excitatory projection neurons in the downstream area, suggesting a basis for direct communication from dorsal hippocampal networks involved in spatial memory and navigation to neocortical networks involved in diverse aspects of sensorimotor integration and motor control. SIGNIFICANCE STATEMENT Corticocortical pathways interconnect cortical areas extensively, but the cellular connectivity in these pathways remains largely uncharacterized. Here, we

  2. A randomized comparison of daunorubicin 90 mg/m2 vs 60 mg/m2 in AML induction

    DEFF Research Database (Denmark)

    Burnett, A. K.; Russell, N. H.; Hills, R. K.

    2015-01-01

    Modifying induction therapy in acute myeloid leukemia (AML) may improve the remission rate and reduce the risk of relapse, thereby improving survival. Escalation of the daunorubicin dose to 90 mg/m(2) has shown benefit for some patient subgroups when compared with a dose of 45 mg/m(2), and has been...... = .15). In an exploratory subgroup analysis, there was no subgroup that showed significant benefit, although there was a significant interaction by FLT3 ITD mutation. This trial is registered at http://www.isrctn.com as #ISRCTN55675535....

  3. Radiation induced genetic variability studies in M2 and F2M2 generation in chilli (Capsicum annum L.)

    International Nuclear Information System (INIS)

    Rangaiah, S.; Manjunath, A.; Naik, Puttarama; Gangappa, E.

    2002-01-01

    Chilli (Capsicum annum L.) is an important commercial crop in India. Mutation breeding is one of the effective tool to create new variability. Since, yield and its component characters show polygenic inheritance, information on amount of heritable portion of variability created through mutation for these characters is needed to use the induced variability for crop improvement. To harness more variability mutation has been superimposed on hybridization in several crops. The present study is undertaken to estimate the genetic variability induced through gamma irradiation for the polygenically inherited productive traits of chilli in M 2 and F 2 M 2 generation

  4. Form-deprivation myopia induces decreased expression of bone morphogenetic protein-2, 5 in guinea pig sclera

    Directory of Open Access Journals (Sweden)

    Qing Wang

    2015-02-01

    Full Text Available AIM: To identify the presence of various bone morphogenetic proteins (BMPs and their receptors in normal sclera of human, rat and guinea pigs, and to determine whether their expression changed with form-deprivation myopia (FDM in guinea pig sclera. METHODS: The expression of BMPs and BMP receptors were detected using reverse transcription polymerase chain reaction (RT-PCR and immunofluorescence. Two-week-old guinea pigs were monocularly form-deprived with a translucent lens. After fourteen days induction of FDM, total RNA was isolated and subjected to RT-PCR to examine the changes of BMPs and BMP receptors in tissues from the posterior sclera. Western blotting analysis was used to investigate their changes in protein levels. RESULTS: Human sclera expressed mRNAs for BMP-2, -4, -5, -7, -RIA, -RIB and BMP-RII. Conversely, rat sclera only expressed mRNA for BMP-7 and BMP-RIB, while the expression of BMPs and BMP receptors in guinea pigs were similar to that of humans. Human sclera also expresses BMP-2, -4, -5,-7 in protein level. Fourteen days after the induction of myopia, significant decreased expressions for BMP-2 and BMP-5 in the posterior sclera of FDM-affected eyes (PCONCLUSION: Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, expressions of BMP-2 and BMP-5 significantly decreased in FDM eyes. This finding indicates that various BMPs as components of the scleral cytokines regulating tissue homeostasis and provide evidence that alterations in the expression of BMP-2 and BMP-5 are associated with sclera remodeling during myopia induction.

  5. Specific Binding of Adamantane Drugs and Direction of their Polar Amines in the Pore of the Influenza M2 Transmembrane Domain in Lipid Bilayers and Dodecylphosphocholine Micelles Determined by NMR Spectroscopy

    Science.gov (United States)

    Cady, Sarah D.; Wang, Jun; Wu, Yibing; DeGrado, William F.; Hong, Mei

    2011-01-01

    The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins. PMID:21381693

  6. Draft Genome Sequence of Bacillus mycoides M2E15, a Strain Isolated from the Endosphere of Potato

    NARCIS (Netherlands)

    Yi, Yanglei; de Jong, Anne; Spoelder, Jan; Elzenga, J Theo M; van Elsas, Jan Dirk; Kuipers, Oscar P

    2016-01-01

    We present the draft genome sequence of Bacillus mycoides M2E15, a bacterium isolated from potato endosphere. Analysis of the 6.08-Mbp draft genome sequence identified 6,386 protein-encoding sequences, including potential plant growth promoting genes. Specifically, genes for proteins involved in

  7. INTERACTION BETWEEN DIFFERENT MOLECULAR FORMS OF IMMUNOGLOBULIN A AND RECOMBINANT DERIVATIVES POLYPEPTIDES OF BAC RECEPTOR PROTEINS FROM GROUP B STREPTOCOCCI

    Directory of Open Access Journals (Sweden)

    A. S. Korzhueva

    2008-01-01

    Full Text Available Abstract. The article concerns interactions between immunoglobulin A and recombinant P6, P7, P8 polypeptides, designed on the basis of externally localized Bac protein of the Group B streptococci, possessing IgA-binding activity.There is a current demand for immunochemical reagents that are strictly specific for IgA, in order to develop antigenic standards for detection of IgA levels in biological fluids, as well as for affinity purification of IgA and its fragments.To analyze an opportunity of the abovementioned application ways for these proteins, a special study was performed to assay an interaction capability of recombinant P6, P7, P8 polypeptides binding to Fc regions of different IgA forms (serum IgA, secretory IgA, subclasses of serum IgA – IgA1, IgA2. Selectivity of ligand binding was specially confirmed.It was found out that, among three presented polypeptides, the structure of recombinant P6 derivative proved to be optimal for IgA-binding ability of Bac protein.Structural features of IgA-binding fragments of Bac protein, i.e., binding site position on the IgA molecule (proximity to epitopes for three monoclonal antibodies, variability of the site structure, as well as resistance of binding site for P6, P7, P8 in IgA molecule against partial disulfide bonds reduction. (Med. Immunol., vol. 10, N 4-5, pp 327-336.

  8. Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

    Directory of Open Access Journals (Sweden)

    Liudmila A Stepanova

    Full Text Available Matrix 2 protein ectodomain (M2e is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek. Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1 and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1 and A/Chicken/Kurgan/05/05 RG (H5N1 to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2 and avian influenza virus (H5N1. Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.

  9. On relating multiple M2 and D2-branes

    International Nuclear Information System (INIS)

    Gran, U.; Nilsson, B.E.W; Petersson, C.

    2008-01-01

    Due to the difficulties of finding superconformal Lagrangian theories for multiple M2-branes, we will in this paper instead focus on the field equations. By relaxing the requirement of a Lagrangian formulation we can explore the possibility of having structure constants f ABC D satisfying the fundamental identity but which are not totally antisymmetric. We exemplify this discussion by making use of an explicit choice of a non-antisymmetric f ABC D constructed from the Lie algebra structure constants f ab c of an arbitrary gauge group. Although this choice of f ABC D does not admit an obvious Lagrangian description, it does reproduce the correct SYM theory for a stack of N D2-branes to leading order in g YM -1 upon reduction and, moreover, it sheds new light on the centre of mass coordinates for multiple M2-branes.

  10. Highly retarded M2 transition in 90Nb

    International Nuclear Information System (INIS)

    Sergeev, V.O.

    2006-01-01

    One studied properties of 2.3 keV transition between 124.67 (4 - ) and 122.37 keV (6 + ) in 90 Nb. The mentioned M2-transition is shown to be retarded by approximately 10 4 times and provided explanations of the mentioned extraordinary high factor of deceleration. One evaluated impurity of E3 possible multipolarity constituent in the mentioned transition (δ 2 [ru

  11. Solar thermal barometer. 12 million M2 installed in europe

    International Nuclear Information System (INIS)

    Anon.

    2002-01-01

    European thermal solar has once again reached its zenith. The difficult times of 1985-1995 are now a thing of the past. The best proof is the very good results of 2001 that show figures in significant progression with respect to those of the year 2000. 1480 320 m 2 . This is now the new record to beat in terms of the annual volume of thermal solar collector installation in the European Union. (author)

  12. Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

    Science.gov (United States)

    Mori, Yu; Adams, Douglas; Hagiwara, Yusuke; Yoshida, Ryu; Kamimura, Masayuki; Itoi, Eiji; Rowe, David W

    2016-11-01

    Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

  13. State-of-the-art Model M-2 Maintenance System

    International Nuclear Information System (INIS)

    Herndon, J.N.; Martin, H.L.; Satterlee, P.E. Jr.; Jelatis, D.G.; Jennrich, C.E.

    1984-04-01

    The Model M-2 Maintenance System is part of an ongoing program within the Consolidated Fuel Reprocessing Program (CFRP) at Oak Ridge National Laboratory (ORNL) to improve remote manipulation technology for future nuclear fuel reprocessing and other remote applications. Techniques, equipment, and guidelines which can improve the efficiency of remote maintenance are being developed. The Model M-2 Maintenance System, installed in the Integrated Equipment Test (IET) Facility at ORNL, provides a complete, integrated remote maintenance system for the demonstration and development of remote maintenance techniques. The system comprises a pair of force-reflecting servomanipulator arms, television viewing, lighting, and auxiliary lifting capabilities, thereby allowing manlike maintenance operations to be executed remotely within the remote cell mockup area in the IET. The Model M-2 Maintenance System incorporates an upgraded version of the proven Central Research Laboratories' Model M servomanipulator. Included are state-of-the-art brushless dc servomotors for improved performance, remotely removable wrist assemblies, geared azimuth drive, and a distributed microprocessor-based digital control system. 5 references, 8 figures

  14. miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Angela M Liu

    Full Text Available In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect, with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122 is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC. Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001. In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2 is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

  15. Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

    Science.gov (United States)

    Mokhtarian, Kobra; Meamar, Ahmad Reza; Khoshmirsafa, Majid; Razmjou, Elham; Masoori, Leila; Khanmohammadi, Majid; Akhlaghi, Lame; Falak, Reza

    2018-01-01

    Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions.

  16. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  17. Plant plasma membrane 14-3-3 proteins differ in solubility and form fusicoccin-dependent complexes

    NARCIS (Netherlands)

    Korthout, H.A.A.J.; de Boer, A.H.

    1998-01-01

    The binding protein for the phytotoxin fusicoccin belongs to the class of highly conserved 14-3-3 proteins. A general principle for the mode of action of 14-3-3 proteins is that they serve as docking clamps in order to facilitate protein interactions. This implies that 14-3-3 proteins may behave

  18. Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice.

    Science.gov (United States)

    Kim, Ahreum; Hur, Yun-Gyoung; Gu, Sunwha; Cho, Sang-Nae

    2017-11-01

    The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis -infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses ( P tuberculosis Beijing/K-strain is frequently isolated from TB patients. Copyright © 2017 American Society for Microbiology.

  19. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    Science.gov (United States)

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  20. The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

    Science.gov (United States)

    Saunders, Keith; Lomonossoff, George P

    2015-01-01

    Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

  1. Kinetics of proton transport into influenza virions by the viral M2 channel.

    Directory of Open Access Journals (Sweden)

    Tijana Ivanovic

    Full Text Available M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

  2. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Sergei; Pakhomov, Alexey A.; Chertkova, Rita V.; Martynov, Vladimir I.; Muslinkina, Liya; Dauter, Zbigniew; Pletnev, Vladimir Z.

    2016-07-13

    The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

  3. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

    Energy Technology Data Exchange (ETDEWEB)

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-09-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  4. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

    Science.gov (United States)

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-01-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  5. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio

    KAUST Repository

    Gomes-Santos, Carina S. S.

    2011-05-19

    Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism. 2011 Gomes-Santos et al.

  6. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

    Science.gov (United States)

    Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

    2013-06-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

  7. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

    Directory of Open Access Journals (Sweden)

    Gale Smith

    Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

  8. Solution NMR structure of the V27A drug resistant mutant of influenza A M2 channel

    Energy Technology Data Exchange (ETDEWEB)

    Pielak, Rafal M. [Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 (United States); Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115 (United States); Chou, James J., E-mail: chou@cmcd.hms.harvard.edu [Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-08

    Research highlights: {yields} This paper reports the structure of the V27A drug resistant mutant of the M2 channel of influenza A virus. {yields} High quality NMR data allowed a better-defined structure for the C-terminal region of the M2 channel. {yields} Using the structure, we propose a proton transfer pathway during M2 proton conduction. {yields} Structural comparison between the wildtype, V27A and S31N variants allowed an in-depth analysis of possible modes of drug resistance. {yields} Distinct feature of the V27A channel pore also provides an explanation for its faster rate of proton conduction. -- Abstract: The M2 protein of influenza A virus forms a proton-selective channel that is required for viral replication. It is the target of the anti-influenza drugs, amantadine and rimantadine. Widespread drug resistant mutants, however, has greatly compromised the effectiveness of these drugs. Here, we report the solution NMR structure of the highly pathogenic, drug resistant mutant V27A. The structure reveals subtle structural differences from wildtype that maybe linked to drug resistance. The V27A mutation significantly decreases hydrophobic packing between the N-terminal ends of the transmembrane helices, which explains the looser, more dynamic tetrameric assembly. The weakened channel assembly can resist drug binding either by destabilizing the rimantadine-binding pocket at Asp44, in the case of the allosteric inhibition model, or by reducing hydrophobic contacts with amantadine in the pore, in the case of the pore-blocking model. Moreover, the V27A structure shows a substantially increased channel opening at the N-terminal end, which may explain the faster proton conduction observed for this mutant. Furthermore, due to the high quality NMR data recorded for the V27A mutant, we were able to determine the structured region connecting the channel domain to the C-terminal amphipathic helices that was not determined in the wildtype structure. The new structural

  9. M2-Branes in N = 3 Harmonic Superspace

    Directory of Open Access Journals (Sweden)

    E. Ivanov

    2010-01-01

    Full Text Available We give a brief account of the recently proposed N = 3 superfield formulation of the N = 6, 3D superconformal theory of Aharony et al (ABJM describing a low-energy limit of the system of multiple M2-branes on the AdS4×S7/Zk background. This formulation is given in harmonic N = 3 superspace and reveals a number of surprising new features. In particular, the sextic scalar potential of ABJM arises at the on-shell component level as the result of eliminating appropriate auxiliary fields, while there is no explicit superpotential at the off-shell superfield level.

  10. Oligomeric forms of the metastasis-related Mts1 (S100A4) protein stimulate neuronal differentiation in cultures of rat hippocampal neurons

    DEFF Research Database (Denmark)

    Novitskaya, V; Grigorian, M; Kriajevska, M

    2000-01-01

    protein family. The oligomeric but not the dimeric form of Mts1 strongly induces differentiation of cultured hippocampal neurons. A mutant with a single Y75F amino acid substitution, which stabilizes the dimeric form of Mts1, is unable to promote neurite extension. Disulfide bonds do not play an essential...

  11. SLiM 2: Flexible, Interactive Forward Genetic Simulations.

    Science.gov (United States)

    Haller, Benjamin C; Messer, Philipp W

    2017-01-01

    Modern population genomic datasets hold immense promise for revealing the evolutionary processes operating in natural populations, but a crucial prerequisite for this goal is the ability to model realistic evolutionary scenarios and predict their expected patterns in genomic data. To that end, we present SLiM 2: an evolutionary simulation framework that combines a powerful, fast engine for forward population genetic simulations with the capability of modeling a wide variety of complex evolutionary scenarios. SLiM achieves this flexibility through scriptability, which provides control over most aspects of the simulated evolutionary scenarios with a simple R-like scripting language called Eidos. An example SLiM simulation is presented to illustrate the power of this approach. SLiM 2 also includes a graphical user interface for simulation construction, interactive runtime control, and dynamic visualization of simulation output, facilitating easy and fast model development with quick prototyping and visual debugging. We conclude with a performance comparison between SLiM and two other popular forward genetic simulation packages. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    DEFF Research Database (Denmark)

    Li, Yiping; Kang, H.N.; Babiuk, L.A.

    2006-01-01

    boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-gamma secreting cells, and cytotoxic T lymphocyte assays...... and shifted the immune response towards Th2-like ones in piglets. CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response......AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without...

  13. Biophysical characterization of the olfactomedin domain of myocilin, an extracellular matrix protein implicated in inherited forms of glaucoma.

    Directory of Open Access Journals (Sweden)

    Susan D Orwig

    Full Text Available Myocilin is an eye protein found in the trabecular extracellular matrix (TEM, within the anatomic region that controls fluid flow. Variants of myocilin, localized to its olfactomedin (OLF domain, have been linked to inherited forms of glaucoma, a disease associated with elevated intraocular pressure. OLF domains have also been implicated in psychiatric diseases and cancers by their involvement in signaling, neuronal growth, and development. However, molecular characterization of OLFs has been hampered by challenges in recombinant expression, a hurdle we have recently overcome for the myocilin OLF domain (myoc-OLF. Here, we report the first detailed solution biophysical characterization of myoc-OLF to gain insight into its structure and function. Myoc-OLF is stable in the presence of glycosaminoglycans, as well as in a wide pH range in buffers with functional groups reminiscent of such glycosaminoglycans. Circular dichroism (CD reveals significant β-sheet and β-turn secondary structure. Unexpectedly, the CD signature is reminiscent of α-chymotrypsin as well as another ocular protein family, the βγ-crystallins. At neutral pH, intrinsic tryptophan fluorescence and CD melts indicate a highly cooperative transition with a melting temperature of ∼55 °C. Limited proteolysis combined with mass spectrometry reveals that the compact core structural domain of OLF consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. The data presented here inform new testable hypotheses for interactions with specific TEM components, and will assist in design of therapeutic agents for myocilin glaucoma.

  14. Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor.

    OpenAIRE

    Moscatelli, D; Joseph-Silverstein, J; Manejias, R; Rifkin, D B

    1987-01-01

    A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator pro...

  15. miR-181a Induces Macrophage Polarized to M2 Phenotype and Promotes M2 Macrophage-mediated Tumor Cell Metastasis by Targeting KLF6 and C/EBPα

    Directory of Open Access Journals (Sweden)

    Jia Bi

    2016-01-01

    Full Text Available Macrophages can acquire a variety of polarization status and functions: classically activated macrophages (M1 macrophages; alternatively activated macrophages (M2 macrophages. However, the molecular basis of the process is still unclear. Here, this study addresses that microRNA-181a (miR-181a is a key molecule controlling macrophage polarization. We found that miR-181a is overexpressed in M2 macrophages than in M1 macrophages. miR-181a expression was decreased when M2 phenotype converted to M1, whereas it increased when M1 phenotype converted to M2. Overexpression of miR-181a in M1 macrophages diminished M1 phenotype expression while promoting polarization to the M2 phenotype. In contrast, knockdown of miR-181a in M2 macrophages promoted M1 polarization and diminished M2 phenotype expression. Mechanistically, Bioinformatic analysis revealed that Kruppel-like factor 6 (KLF6 and CCAAT/enhancer binding protein-α (C/EBPα is a potential target of miR-181a and luciferase assay confirmed that KLF6 and C/EBPα translation is suppressed by miR-181a through interaction with the 3′UTR of KLF6 and C/EBPα mRNA. Further analysis showed that induction of miR-181a suppressed KLF6 and C/EBPα protein expression. Importantly, miR-181a also diminishes M2 macrophages-mediated migration and invasion capacity of tumor cells. Collectively, our results suggest that miR-181a plays a significant role in regulating macrophage polarization through directly target KLF6 and C/EBPα.

  16. Properties of the mitochondrial carrier of adenine-nucleotide after purification. Study of the transport protein under isolated form and reincorporated form in phospho-lipidic vesicles

    International Nuclear Information System (INIS)

    Brandolin, Gerard

    1983-01-01

    The first part of this research thesis addresses the reconstitution of the ADP/ATP transport by incorporation of the specific carrier, isolated in presence of detergent, in phospholipids vesicles. Fundamental properties of the reconstituted transport are identical to that of transport in mitochondria, notably as far as the exchange stoichiometry, the turn over and the transport Km are concerned, as well as the asymmetric orientation of the carrier in the membrane. The second part of this research addresses the study of interactions of specific ligands with the ADP/ATP transport protein in presence of detergent. The study of the variations of the intrinsic fluorescence of the isolated ADP/ATP carrier highlights conformational changes exclusively induced by the presence of transportable nucleotides which are modulated in a different manner by carboxy-atractyloside or bongkrekic acid. Moreover, by using the isolated protein, a detailed analysis of binding parameters of fluorescent analogues of ATP is reported [fr

  17. Supersymmetric states in M5/M2 CFTs

    International Nuclear Information System (INIS)

    Bhattacharyya, Sayantani; Minwalla, Shiraz

    2007-01-01

    We propose an exact, finite N formula for the partition function over 1/4 th BPS states in the conformal field theory on the world volume of N coincident M5 branes, and 1/8 th BPS states in the theory of N conincident M2 branes. We obtain our partition function by performing the radial quantization of the Coulomb Branches of these theories and rederive the same formula from the quantization of supersymmetric giant and dual giant gravitons in AdS 7 x S 4 and AdS 4 x S 7 . Our partition function is qualitatively similar to the analogous quantity in N = 4 Yang Mills. It reduces to the sum over supersymmetric multi gravitons at low energies, but deviates from this supergravity formula at energies that scale like a positive power of N

  18. TREAT experiment M2 post-test examination

    International Nuclear Information System (INIS)

    Holland, J.W.; Teske, G.M.; Florek, J.C.

    1986-01-01

    Transient Reactor Test (TREAT) Facility experiment M2 was performed to evaluate the transient behavior of metal-alloy fuel under accident conditions to investigate the inherent safety features of the fuel in integral fast reactor (IFR) system designs. Objectives were to obtain early information on the key fuel behavior characteristics at transient overpower (TOP) conditions in metal-fueled fast reactors; namely, margin to cladding breach and extent of axial self-extrusion of fuel within intact cladding. The onset of cladding breaching depends on fuel/cladding eutectic formation, as well as cladding pressurization and melting. Driving forces for fuel extrusion are fission gas, liquid sodium, and volatile fission products trapped within the fuel matrix. The post-test examination provided data essential for correctly modeling fuel behavior in accident codes

  19. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    Science.gov (United States)

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  20. 13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

    International Nuclear Information System (INIS)

    Siemer, Ansgar B.; Ritter, Christiane; Steinmetz, Michel O.; Ernst, Matthias; Riek, Roland; Meier, Beat H.

    2006-01-01

    The partial 15 N and 13 C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional 13 C-- 13 C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional 15 N-- 13 C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues

  1. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide

    International Nuclear Information System (INIS)

    Genin, Marie; Clement, Francois; Fattaccioli, Antoine; Raes, Martine; Michiels, Carine

    2015-01-01

    Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis

  2. Microwave and magnetic (M2 proteomics of a mouse model of mild traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Teresa M. Evans

    2014-06-01

    Full Text Available Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI. However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave and magnetic (M2 proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP and p < 0.05 for myelin associated glycoprotein (MAG. This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2 and neurofilament light (NEFL expression at 30 days post-injury were directly related to grip strength (p < 0.05. While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M2 proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment.

  3. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2013-04-01

    Full Text Available Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer.

  4. Characterization of structural proteins of hirame rhabdovirus, HRV

    Science.gov (United States)

    Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James; Ahne, Winfried; Kimura, Takahisa

    1991-01-01

    Structural proteins of hirame rhabdovirus (HRV) were analyzed by SDS-polyacrylarnide gel electrophoresis, western blotting, 2-dimensional gel electrophoresis, and Triton X-100 treatment. Purified HRV virions were composed of: polymerase (L), glycoprotein (G), nucleoprotein (N), and 2 matrix proteins (M1 and M2). Based upon their relative mobilities, the estimated molecular weights of the proteins were: L, 156 KDa; G, 68 KDa; N, 46.4 KDa; M1, 26.4 KDa; and M2, 19.9 KDa. The electrophorehc pattern formed by the structural proteins of HRV was clearly different from that formed by pike fry rhabdovirus, spring viremia of carp virus, eel virus of America, and eel virus European X which belong to the Vesiculovirus genus; however, it resembled the pattern formed by structural proteins of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) which are members of the Lyssavirus genus. Among HRV, IHNV, and VHSV, differences were observed in the relative mobilities of the G, N, M1, and M2 proteins. Western blot analysis revealed that the G. N, and M2 proteins of HRV shared antigenic determinants with IHNV and VHSV, but not with any of the 4 fish vesiculoviruses tested. Cross-reactions between the M1 proteins of HRV, IHNV, or VHSV were not detected in this assay. Two-dimensional gel electrophoresis was used to show that HRV differed from IHNV or VHSV in the isoelectric point (PI) of the M1 and M2 proteins. In this system, 2 forms of the M1 protein of HRV and IHNV were observed.These subspecies of M1 had the same relative mobility but different p1 values. Treatment of purified virions with 2% Triton X-100 in Tris buffer containing NaCl removed the G, M1, and M2 proteins of IHNV, but HRV virions were more stable under these conditions.

  5. Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

    Science.gov (United States)

    Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

    2016-01-01

    Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were

  6. An M2e-based synthetic peptide vaccine for influenza A virus confers heterosubtypic protection from lethal virus challenge.

    Science.gov (United States)

    Ma, Ji-Hong; Yang, Fu-Ru; Yu, Hai; Zhou, Yan-Jun; Li, Guo-Xin; Huang, Meng; Wen, Feng; Tong, Guangzhi

    2013-07-09

    Vaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses. Our results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds' adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus. Based on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.

  7. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Ostreolysin A/Pleurotolysin B and Equinatoxins: Structure, Function and Pathophysiological Effects of These Pore-Forming Proteins

    Directory of Open Access Journals (Sweden)

    Robert Frangež

    2017-04-01

    Full Text Available Acidic ostreolysin A/pleurotolysin B (OlyA/PlyB, formerly known as ostreolysin (Oly, and basic 20 kDa equinatoxins (EqTs are cytolytic proteins isolated from the edible mushroom Pleurotus ostreatus and the sea anemone Actinia equina, respectively. Both toxins, although from different sources, share many similar biological activities: (i colloid-osmotic shock by forming pores in cellular and artificial membranes enriched in cholesterol and sphingomyelin; (ii increased vascular endothelial wall permeability in vivo and perivascular oedema; (iii dose-dependent contraction of coronary vessels; (iv haemolysis with pronounced hyperkalaemia in vivo; (v bradycardia, myocardial ischemia and ventricular extrasystoles accompanied by progressive fall of arterial blood pressure and respiratory arrest in rodents. Both types of toxins are haemolytic within nanomolar range concentrations, and it seems that hyperkalaemia plays an important role in toxin cardiotoxicity. However, it was observed that the haemolytically more active EqT III is less toxic than EqT I, the most toxic and least haemolytic EqT. In mice, EqT II is more than 30 times more toxic than OlyA/PlyB when applied intravenously. These observations imply that haemolysis with hyperkalaemia is not the sole cause of the lethal activity of both toxins. Additional mechanisms responsible for lethal action of the two toxins are direct effects on heart, coronary vasoconstriction and related myocardial hypoxia. In this review, we appraise the pathophysiological mechanisms related to the chemical structure of OlyA/PlyB and EqTs, as well as their toxicity.

  9. Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms

    International Nuclear Information System (INIS)

    Reis, Marcelo Augusto dos; Saraiva, Antonio Marcos; Santos, Marcelo Leite dos; Souza, Anete Pereira de; Aparicio, Ricardo

    2012-01-01

    The crystallization and preliminary analysis of two crystal forms of survival protein E from X. fastidiosa are reported. The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, β = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, β = 94.76°

  10. Microglia M2A Polarization as Potential Link between Food Allergy and Autism Spectrum Disorders

    Directory of Open Access Journals (Sweden)

    Hans O. Kalkman

    2017-12-01

    Full Text Available Atopic diseases are frequently co-morbid with autism spectrum disorders (ASD. Allergic responses are associated with an activation of mast cells, innate lymphoid cells, and Th2 cells. These cells produce type-2 cytokines (IL4 and IL13, which stimulate microglia and macrophages to adopt a phenotype referred to as ‘alternative activation’ or ‘M2A’. M2A-polarized macrophages and microglia play a physiological role in tissue repair by secreting growth factors such as brain-derived neurotrophic factor (BDNF and insulin-like growth factor-1. In ASD there is evidence for increased type-2 cytokines, microglia activation, M2A polarization, and increased levels of growth factors. In neurons, these growth factors drive a signal transduction pathway that leads to activation of the enzyme mammalian Target of Rapamycin (mTOR, and thereby to the inhibition of autophagy. Activation of mTOR is an effect that is also common to several of the genetic forms of autism. In the central nervous system, redundant synapses are removed via an autophagic process. Activation of mTOR would diminish the pruning of redundant synapses, which in the context of ASD is likely to be undesired. Based on this line of reasoning, atopic diseases like food allergy, eczema or asthma would represent risk factors for autism spectrum disorders.

  11. Study on surface modification of M2 steel induced by Cu ions and Al ions implantation

    International Nuclear Information System (INIS)

    Wang Chao; Liu Zhengmin

    2001-01-01

    Changes of surface hardness and wear resistances in M2 type steel implanted by Cu Al ions were reported. The dependence of surface strengthening on ion species and dose was studied by X-ray diffraction (XRD) and Rutherford Backscattering Spectroscopy (RBS) for microhardness and wear resistances measurement. It is shown that both hardness and wear resistance increases apparently after ion implantation. XRD analysis indicates that different phases formed after Al Cu ions implanted. It is also suggested that Cu, Al ions have different role in surface strengthening

  12. Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

    NARCIS (Netherlands)

    Bossers, A.; Belt, P.B.G.M.; Raymond, G.J.; Caughey, B.; Vries, de R.; Smits, M.

    1997-01-01

    Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system.

  13. Natural loss-of-function mutation of myeloid differentiation protein 88 disrupts its ability to form Myddosomes

    NARCIS (Netherlands)

    Nagpal, K.; Plantinga, T.S.; Sirois, C.M.; Monks, B.G.; Latz, E.; Netea, M.G.; Golenbock, D.T.

    2011-01-01

    Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a

  14. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Directory of Open Access Journals (Sweden)

    Sylvaine Boissinot

    Full Text Available Cucurbit aphid-borne yellows virus (CABYV is a polerovirus (Luteoviridae family with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT. Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT* incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  15. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Science.gov (United States)

    Boissinot, Sylvaine; Erdinger, Monique; Monsion, Baptiste; Ziegler-Graff, Véronique; Brault, Véronique

    2014-01-01

    Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  16. Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh

    Energy Technology Data Exchange (ETDEWEB)

    Boeckmann, Anja [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France)], E-mail: a.bockmann@ibcp.fr; Lange, Adam [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Galinier, Anne [Institut de Biologie Structurale et Microbiologie, C.N.R.S UPR 9043 (France); Luca, Sorin [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Giraud, Nicolas; Juy, Michel [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Heise, Henrike [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Montserret, Roland; Penin, Francois [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Baldus, Marc [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany)], E-mail: maba@mpibpc.mpg.de

    2003-12-15

    Solid state NMR sample preparation and resonance assignments of the U-[{sup 13}C,{sup 15}N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all {sup 15}N, {sup 13}C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

  17. Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

    Science.gov (United States)

    Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

    2010-03-01

    The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

  18. A Polypyrimidine Tract Binding Protein, Pumpkin RBP50, Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex[W

    Science.gov (United States)

    Ham, Byung-Kook; Brandom, Jeri L.; Xoconostle-Cázares, Beatriz; Ringgold, Vanessa; Lough, Tony J.; Lucas, William J.

    2009-01-01

    RNA binding proteins (RBPs) are integral components of ribonucleoprotein (RNP) complexes and play a central role in RNA processing. In plants, some RBPs function in a non-cell-autonomous manner. The angiosperm phloem translocation stream contains a unique population of RBPs, but little is known regarding the nature of the proteins and mRNA species that constitute phloem-mobile RNP complexes. Here, we identified and characterized a 50-kD pumpkin (Cucurbita maxima cv Big Max) phloem RNA binding protein (RBP50) that is evolutionarily related to animal polypyrimidine tract binding proteins. In situ hybridization studies indicated a high level of RBP50 transcripts in companion cells, while immunolocalization experiments detected RBP50 in both companion cells and sieve elements. A comparison of the levels of RBP50 present in vascular bundles and phloem sap indicated that this protein is highly enriched in the phloem sap. Heterografting experiments confirmed that RBP50 is translocated from source to sink tissues. Collectively, these findings established that RBP50 functions as a non-cell-autonomous RBP. Protein overlay, coimmunoprecipitation, and cross-linking experiments identified the phloem proteins and mRNA species that constitute RBP50-based RNP complexes. Gel mobility-shift assays demonstrated that specificity, with respect to the bound mRNA, is established by the polypyrimidine tract binding motifs within such transcripts. We present a model for RBP50-based RNP complexes within the pumpkin phloem translocation stream. PMID:19122103

  19. Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains

    NARCIS (Netherlands)

    Domeradzka, Natalia E.; Werten, Marc W.T.; Vries, de Renko; Wolf, de Frits A.

    2016-01-01

    Background: Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting

  20. Report ETSI M2M-14bis JTC March 2011 meeting

    NARCIS (Netherlands)

    Keesmaat, N.W.

    2011-01-01

    The ETSI Technical Committee (TC) M2M focuses on the development of a generic M2M architecture and underlying protocols suitable for a wide range of M2M environments. Whereas in the past effort has been put in the development of several use cases, the focus of this M2M 14bis meeting – held in Sophia

  1. Muscle-Derived Proteins as Serum Biomarkers for Monitoring Disease Progression in Three Forms of Muscular Dystrophy

    Science.gov (United States)

    Burch, Peter M.; Pogoryelova, Oksana; Goldstein, Richard; Bennett, Donald; Guglieri, Michela; Straub, Volker; Bushby, Kate; Lochmüller, Hanns; Morris, Carl

    2015-01-01

    Abstract Background: Identifying translatable, non-invasive biomarkers of muscular dystrophy that better reflect the disease pathology than those currently available would aid the development of new therapies, the monitoring of disease progression and the response to therapy. Objective: The goal of this study was to evaluate a panel of serum protein biomarkers with the potential to specifically detect skeletal muscle injury. Method: Serum concentrations of skeletal troponin I (sTnI), myosin light chain 3 (Myl3), fatty acid binding protein 3 (FABP3) and muscle-type creatine kinase (CKM) proteins were measured in 74 Duchenne muscular dystrophy (DMD), 38 Becker muscular dystrophy (BMD) and 49 Limb-girdle muscular dystrophy type 2B (LGMD2B) patients and 32 healthy controls. Results: All four proteins were significantly elevated in the serum of these three muscular dystrophy patient populations when compared to healthy controls, but, interestingly, displayed different profiles depending on the type of muscular dystrophy. Additionally, the effects of patient age, ambulatory status, cardiac function and treatment status on the serum concentrations of the proteins were investigated. Statistical analysis revealed correlations between the serum concentrations and certain clinical endpoints including forced vital capacity in DMD patients and the time to walk ten meters in LGMD2B patients. Serum concentrations of these proteins were also elevated in two preclinical models of muscular dystrophy, the mdx mouse and the golden-retriever muscular dystrophy dog. Conclusions: These proteins, therefore, are potential muscular dystrophy biomarkers for monitoring disease progression and therapeutic response in both preclinical and clinical studies. PMID:26870665

  2. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  3. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

    Science.gov (United States)

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R; Kehn-Hall, Kylene; Omichinski, James G

    2015-05-12

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

  4. Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

    International Nuclear Information System (INIS)

    Gabrielsen, Mads; Rey-Burusco, M. Florencia; Griffiths, Kate; Roe, Andrew J.; Cooper, Alan; Smith, Brian O.; Kennedy, Malcolm W.; Corsico, Betina

    2012-01-01

    Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions. Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning

  5. Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H] hydrocortisone

    International Nuclear Information System (INIS)

    Voigt, J.; Grote, H.; Sekeris, C.E.

    1981-01-01

    After administration of [ 3 H] hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [ 3 H] hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5-60 min after administration of biologically active doses of hydrocortisone. 3xi,11β,17α,20xi, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all the cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in the nuclear envelope. (orig.)

  6. Electrochemical, atomic force microscopy and infrared reflection absorption spectroscopy studies of pre-formed mussel adhesive protein films on carbon steel for corrosion protection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fan, E-mail: fanzhang@kth.se [KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Chemistry, Div. of Surface and Corrosion Science, Drottning Kristinas vaeg.51, SE-100 44 Stockholm (Sweden); Pan, Jinshan [KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Chemistry, Div. of Surface and Corrosion Science, Drottning Kristinas vaeg.51, SE-100 44 Stockholm (Sweden); Claesson, Per Martin [KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Chemistry, Div. of Surface and Corrosion Science, Drottning Kristinas vaeg.51, SE-100 44 Stockholm (Sweden); Institute for Surface Chemistry, P.O. Box 5607, SE-114 86 Stockholm (Sweden); Brinck, Tore [KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Physical Chemistry, Division of Physical Chemistry, Teknikringen 36, SE-10044 Stockholm (Sweden)

    2012-10-01

    Electrochemical measurements, in situ and ex situ atomic force microscopy (AFM) experiments and infrared reflection absorption spectroscopy (IRAS) analysis were performed to investigate the formation and stability as well as corrosion protection properties of mussel adhesive protein (Mefp-1) films on carbon steel, and the influence of cross-linking by NaIO{sub 4} oxidation. The in situ AFM measurements show flake-like adsorbed protein aggregates in the film formed at pH 9. The ex situ AFM images indicate multilayer-like films and that the film becomes more compact and stable in NaCl solution after the cross-linking. The IRAS results reveal the absorption bands of Mefp-1 on carbon steel before and after NaIO{sub 4} induced oxidation of the pre-adsorbed protein. Within a short exposure time, a certain corrosion protection effect was noted for the pre-formed Mefp-1 film in 0.1 M NaCl solution. Cross-linking the pre-adsorbed film by NaIO{sub 4} oxidation significantly enhanced the protection efficiency by up to 80%. - Highlights: Black-Right-Pointing-Pointer Mussel protein was tested as 'green' corrosion protection strategy for steel. Black-Right-Pointing-Pointer At pH 9, the protein adsorbs on carbon steel and forms a multilayer-like film. Black-Right-Pointing-Pointer NaIO{sub 4} leads to structural changes and cross-linking of the protein film. Black-Right-Pointing-Pointer Cross-linking results in a dense and compact film with increased stability. Black-Right-Pointing-Pointer Cross-linking of preformed film significantly enhances the corrosion protection.

  7. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  8. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  9. M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

    Science.gov (United States)

    Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

    2013-01-01

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

  10. Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer

    Directory of Open Access Journals (Sweden)

    Gopinath Prakasam

    2018-02-01

    Full Text Available Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

  11. Anti-Inflammatory Strategy for M2 Microglial Polarization Using Retinoic Acid-Loaded Nanoparticles

    Directory of Open Access Journals (Sweden)

    Marta Machado-Pereira

    2017-01-01

    Full Text Available Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2. RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state, promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases.

  12. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the

  13. Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

    Science.gov (United States)

    Scott, Evan A.; Nichols, Michael D.; Cordova, Lee H.; George, Brandon J.; Jun, Young-Shin; Elbert, Donald L.

    2008-01-01

    Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically-active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating. PMID:18771802

  14. First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form.

    Science.gov (United States)

    de Rocquigny, H; Ficheux, D; Gabus, C; Fournié-Zaluski, M C; Darlix, J L; Roques, B P

    1991-10-31

    The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

  15. The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

    Science.gov (United States)

    Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia; Zhang, Bo; Mapolelo, Daphne T; Randeniya, Sajini; Holland, Ashley A; Johnson, Michael K; Outten, Caryn E

    2016-12-13

    Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

  16. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

    Energy Technology Data Exchange (ETDEWEB)

    Krasnoslobodtsev, Alexey V., E-mail: akrasnos@unomaha.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Department of Physics, University of Nebraska Omaha, Omaha, NE 68182 (United States); Deckert-Gaudig, Tanja [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Zhang, Yuliang [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Deckert, Volker [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Institute for Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena (Germany); Lyubchenko, Yuri L., E-mail: ylyubchenko@unmc.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States)

    2016-06-15

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. - Highlights: • Amyloid polymorphs were characterized by AFM and TERS. • A mixture of peptide secondary structures in fibrils were identified using TERS. • TERS recognizes packing arrangement (parallel versus antiparallel) of peptides. • TERS is a powerful tool for high resolution structural analysis of fibrils.

  17. Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.

    Science.gov (United States)

    Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

    2016-06-10

    Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing. We constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P. pastoris with a purified protein yield of more than 2 g L(-1) of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM. This article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.

  18. Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

    Science.gov (United States)

    Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

    2017-01-01

    Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

  19. Use of M2e ELISAs for longitudinal surveillance of commercial poultry in Indonesia vaccinated against highly pathogenic avian influenza.

    Science.gov (United States)

    Wibowo, Michael Haryadi; Tarigan, Simson; Sumarningsih; Artanto, Sidna; Indriani, Risa; Anggoro, Dito; Putra, Cahyaditya Pratama; Idris, Syafrison; Untari, Tri; Asmara, Widya; Tabbu, Charles Rangga; Ignjatovic, Jagoda

    2017-11-01

    In countries where highly pathogenic avian influenza virus (HPAIV) H5N1 is endemic and controlled by vaccination, post-vaccination serological monitoring is essential to differentiate vaccinated poultry from those that are infected. The objectives of this study were to validate two experimental ELISAs that detect antibodies raised against the M2e protein of avian influenza virus that can be used for DIVA purposes. Results from the sM2e and tM2e ELISAs were compared with other conventional tests for the detection of H5N1influenza virus (virus isolation and RT-PCR) using samples collected from 16 commercial flocks in Indonesia. These comprised vaccinated layers aged between 18 and 68 weeks old that were sampled at ten-weekly intervals. A small number of sera were positive in sM2e and tM2e ELISA, 14 (0.6%) and 17 (0.7%) respectively, with low OD 420 (0.1-0.3), but only 4 sera were positive in both tests. At the flock level, the incidence of M2e positive sera was low (4%), well below previously established minimum of 40% for an HPAIV H5N1-infected flock. Conventional M and H5 gene RT-PCRs indicated that none of 16 flocks were infected at any time during the study. No virus was isolated from any of the 480 pooled swab samples, except from one, for which the combined data analysis suggest to be the result of a laboratory cross-contamination. Clinical disease, mortalities or reduction in production performance, indicative of field H5N1 challenge, were not observed either in any of the flocks. Birds from two surveyed flocks, challenged in the laboratory with an Indonesian HPAIV H5N1 developed M2e antibodies in 50% and 55% of surviving birds with OD 420 in the range of 0.35-1.47 in tM2e ELISA, confirming the validity of the criteria established for use of M2e ELISA for DIVA purposes. Overall these results showed that the tM2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 infections in vaccinated commercial poultry. Copyright © 2017 Elsevier B

  20. An alignment-free method to find similarity among protein sequences via the general form of Chou's pseudo amino acid composition.

    Science.gov (United States)

    Gupta, M K; Niyogi, R; Misra, M

    2013-01-01

    In this paper, we propose a method to create the 60-dimensional feature vector for protein sequences via the general form of pseudo amino acid composition. The construction of the feature vector is based on the contents of amino acids, total distance of each amino acid from the first amino acid in the protein sequence and the distribution of 20 amino acids. The obtained cosine distance metric (also called the similarity matrix) is used to construct the phylogenetic tree by the neighbour joining method. In order to show the applicability of our approach, we tested it on three proteins: 1) ND5 protein sequences from nine species, 2) ND6 protein sequences from eight species, and 3) 50 coronavirus spike proteins. The results are in agreement with known history and the output from the multiple sequence alignment program ClustalW, which is widely used. We have also compared our phylogenetic results with six other recently proposed alignment-free methods. These comparisons show that our proposed method gives a more consistent biological relationship than the others. In addition, the time complexity is linear and space required is less as compared with other alignment-free methods that use graphical representation. It should be noted that the multiple sequence alignment method has exponential time complexity.

  1. M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Jesús Cosín-Roger

    Full Text Available Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in

  2. A stringy origin of M2 brane Chern-Simons theories

    International Nuclear Information System (INIS)

    Aganagic, Mina

    2010-01-01

    We show that string duality relates M-theory on a local Calabi-Yau fourfold singularity X 4 to type IIA string theory on a Calabi-Yau threefold X 3 fibered over a real line, with RR 2-form fluxes turned on. The RR flux encodes how the M-theory circle is fibered over the IIA geometry. The theories on N D2 branes probing X 3 are the well-known quiver theories with N=2 supersymmetry in three dimensions. We show that turning on fluxes, and fibering the X 3 over a direction transverse to the branes, corresponds to turning on N=2 Chern-Simons couplings. String duality implies that, in the strong coupling limit, the N D2 branes on X 3 in this background become N M2 branes on X 4 . This provides a string theory derivation for the recently conjectured description of the M2 brane theories on Calabi-Yau fourfolds in terms of N=2 quiver Chern-Simons theories. We also provide a new N=2 Chern-Simons theory dual to AdS 4 xQ 1,1,1 . Type IIA/M-theory duality also relates IIA string theory on X 3 with only the RR fluxes turned on, to M-theory on a G 2 holonomy manifold. We show that this implies that the N M2 branes probing the G 2 manifold are described by the quiver Chern-Simons theory originating from the D2 branes probing X 3 , except that now Chern-Simons terms preserve only N=1 supersymmetry in three dimensions.

  3. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export*

    Science.gov (United States)

    Port, Sarah A.; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H.

    2016-01-01

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. PMID:27613868

  4. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    Science.gov (United States)

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. M/sub r/ 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Moscatelli, D.; Joseph-Silverstein, J.; Manejias, R.; Rifkin, D.B.

    1987-01-01

    A M/sub r/ 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig grain along with the typical M/sub r/ 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The M/sub r/ 25,000 form, like the M/sub r/ 18,000 form was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The M/sub r/ 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentration of 0.1-10 ngml, the same range that was effective for guinea pig and human M/sub r/ 18,000 bFGFs. The binding of human 125 I-labeled bFGF to baby hamster kidney cells is inhibited equally by the M/sub r/ 25,000 guinea pig protein and the M/sub r/ 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the M/sub r/ 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the M/sub r/ 25,000 and M/sub r/ 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the M/sub r/ 25,000 guinea pig bFGF was converted to a M/sub r/ 18,000 protein. These results show that the two molecules are closely related and suggest that the M/sub r/ 25,000 protein shares substantial homology with the M/sub r/ 18,000 bFGF

  6. A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions

    International Nuclear Information System (INIS)

    Szajner, Patricia; Jaffe, Howard; Weisberg, Andrea S.; Moss, Bernard

    2004-01-01

    Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature

  7. Micrometer and nanometer scale photopatterning of proteins on glass surfaces by photo-degradation of films formed from oligo(ethylene glycol) terminated silanes.

    Science.gov (United States)

    Tizazu, Getachew; el Zubir, Osama; Patole, Samson; McLaren, Anna; Vasilev, Cvetelin; Mothersole, David J; Adawi, Ali; Hunter, C Neil; Lidzey, David G; Lopez, Gabriel P; Leggett, Graham J

    2012-12-01

    Exposure of films formed by the adsorption of oligo(ethylene glycol) (OEG) functionalized trichlorosilanes on glass to UV light from a frequency-doubled argon ion laser (244 nm) causes photodegradation of the OEG chain. Although the rate of degradation is substantially slower than for monolayers of OEG terminated thiolates on gold, it is nevertheless possible to form micrometer-scale patterns by elective adsorption of streptavidin to exposed regions. A low density of aldehyde functional groups is produced, and this enables derivatization with nitrilotriacetic acid via an amine linker. Complexation with nickel enables the site-specific immobilization of histidine-tagged yellow and green fluorescent proteins. Nanometer-scale patterns may be fabricated using a Lloyd's mirror interferometer, with a sample and mirror set at right angles to each other. At low exposures, partial degradation of the OEG chains does not remove the protein-resistance of the surface, even though friction force microscopy reveals the formation of patterns. At an exposure of ca. 18 J cm(-2), the modified regions became adhesive to proteins in a narrow region ca. 30 nm (λ/8) wide. As the exposure is increased further the lines quickly broaden to ca. 90 nm. Adjustment of the angle between the sample and mirror enables the fabrication of lines of His-tagged green fluorescent protein at a period of 340 nm that could be resolved using a confocal microscope.

  8. Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hideshi, E-mail: h-yokoya@u-shizuoka-ken.ac.jp [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Tsuruta, Osamu; Akao, Naoya; Fujii, Satoshi [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Structures of a metal-bound Helicobacter pylori neutrophil-activating protein were determined. Black-Right-Pointing-Pointer Two zinc ions were tetrahedrally coordinated by ferroxidase center (FOC) residues. Black-Right-Pointing-Pointer Two cadmium ions were coordinated in a trigonal-bipyramidal and octahedral manner. Black-Right-Pointing-Pointer The second metal ion was more weakly coordinated than the first at the FOC. Black-Right-Pointing-Pointer A zinc ion was found in one negatively-charged pore suitable as an ion path. -- Abstract: Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn{sup 2+}- or Cd{sup 2+}-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.

  9. Development and evaluation of intelligent machine tools based on knowledge evolution in M2M environment

    International Nuclear Information System (INIS)

    Kim, Dong Hoon; Song, Jun Yeob; Lee, Jong Hyun; Cha, Suk Keun

    2009-01-01

    In the near future, the foreseen improvement in machine tools will be in the form of a knowledge evolution-based intelligent device. The goal of this study is to develop intelligent machine tools having knowledge-evolution capability in Machine to Machine (M2M) wired and wireless environment. The knowledge evolution-based intelligent machine tools are expected to be capable of gathering knowledge autonomously, producing knowledge, understanding knowledge, applying reasoning to knowledge, making new decisions, dialoguing with other machines, etc. The concept of the knowledge-evolution intelligent machine originated from the process of machine control operation by the sense, dialogue and decision of a human expert. The structure of knowledge evolution in M2M and the scheme for a dialogue agent among agent-based modules such as a sensory agent, a dialogue agent and an expert system (decision support agent) are presented in this paper, and work-offset compensation from thermal change and recommendation of cutting condition are performed on-line for knowledge-evolution verification

  10. Preliminary heat treatment of 4KhM2Fch die steel

    International Nuclear Information System (INIS)

    Leonidov, V.M.; Berezkin, Y.A.; Nikitenko, E.V.

    1986-01-01

    To improve the machinability and preparation of the structure for hardening, die steels are given a preliminary treatment which provides a reduction in hardness as a result of separation in the structure of the carbide and ferrite phases, coagulation of the carbides, and acquisition by them of a granular form and also the obtaining of fine grains and a uniform distribution of the structural constituents. The microstructure was evaluated after etching in 4% nital on an MIM-8M microscope. The 4KhM2Fch steel was given a preliminary heat treatment of normalize and anneal. It was concluded that for 4KhM2Fch steel a preliminary heat treatment of normalizing from 950 0 C with a hold of 1.5-2 h, annealing at 750-760 0 C with a hold of 2-3 h, cooling to the isothermal temperature of 670-680 0 C with a hold of 3-4 h, and further air cooling is recommended. The structure after such a heat is granular pearlite with a rating 1-2 and a hardness of 220-250 HB

  11. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

    Science.gov (United States)

    Leser, George P; Lamb, Robert A

    2017-05-01

    Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

  12. Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils.

    Science.gov (United States)

    Araman, Can; Thompson, Robert E; Wang, Siyao; Hackl, Stefanie; Payne, Richard J; Becker, Christian F W

    2017-09-01

    The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179-231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

  13. HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

    Science.gov (United States)

    De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

    2017-09-01

    Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Thermostability of Multidomain Proteins: Elongation Factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and Their Chimeric Forms

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Hůlková, Marta; Maloň, Petr; Kepková, M.; Jonák, Jiří

    2004-01-01

    Roč. 13, č. 1 (2004), s. 89-99 ISSN 0961-8368 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/98/0863; GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : elongation factor EF-Tu, thermostability, chimeric protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.116, year: 2004

  15. Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins.

    Science.gov (United States)

    Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

    2016-11-01

    Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2.

  16. Characterization of changes in composition and function of erythrocyte membrane proteins in patients with bone marrow form of acute radiation sickness

    International Nuclear Information System (INIS)

    Li Jinying; Wei Shanjian; Hu Xiaojian

    1998-01-01

    Objective: The delayed effect of radiation on erythrocyte membrane protein, the composition and function of the membrane proteins in five patients with bone marrow form of acute radiation sickness (ARS) were follow up at six years after the Shanghai 60 Co irradiation accident. Methods: Percoll centrifugation, SDS-polyacrylamide gel electrophoresis, and analysis of NO 2 - transport rate and DIDS inhibition rate were performed. Results: The injuries of the membrane proteins induced by radiation, characterized by reduced content of band 8 and declined anion transport function of band 3 protein remained the same as initially observed. The further study showed that the inhibition of DIDS on the anion transport of the ARS erythrocytes was decreased and the transport time for NO 2 - by band 3 was significantly prolonged in younger erythrocytes than those in middle-or old-aged cells. Conclusion: It is suggested that the radiation damage to erythrocyte membrane proteins might occur at the stage of erythropoiesis in bone marrow. The exo-facial site in band 3 may be changed after radiation, which could result in the abnormalities in anion transport. It is believed that the aging of erythrocytes might be present in advanced stage of ARS

  17. Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone.

    Science.gov (United States)

    Alvarez, Carlos; Ros, Uris; Valle, Aisel; Pedrera, Lohans; Soto, Carmen; Hervis, Yadira P; Cabezas, Sheila; Valiente, Pedro A; Pazos, Fabiola; Lanio, Maria E

    2017-10-01

    Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure-function relationship, the protein-membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin-membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein-lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts-cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Sts-based vaccine platforms.

  18. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    International Nuclear Information System (INIS)

    Valdes, Iris; Bernardo, Lidice; Gil, Lazaro; Pavon, Alekis; Lazo, Laura; Lopez, Carlos; Romero, Yaremis; Menendez, Ivon; Falcon, Viviana; Betancourt, Lazaro; Martin, Jorge; Chinea, Glay; Silva, Ricardo; Guzman, Maria G.; Guillen, Gerardo; Hermida, Lisset

    2009-01-01

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4 + and CD8 + cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  19. A Protein Synthesis and Nitric Oxide-Dependent Presynaptic Enhancement in Persistent Forms of Long-Term Potentiation

    Science.gov (United States)

    Johnstone, Victoria P. A.; Raymond, Clarke R.

    2011-01-01

    Long-term potentiation (LTP) is an important process underlying learning and memory in the brain. At CA3-CA1 synapses in the hippocampus, three discrete forms of LTP (LTP1, 2, and 3) can be differentiated on the basis of maintenance and induction mechanisms. However, the relative roles of pre- and post-synaptic expression mechanisms in LTP1, 2,…

  20. Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

    Science.gov (United States)

    Zajc, Tajana; Suban, Dejan; Rajković, Jelena; Dolenc, Iztok

    2011-02-01

    The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. M2Lite: An Open-source, Light-weight, Pluggable and Fast Proteome Discoverer MSF to mzIdentML Tool.

    Science.gov (United States)

    Aiyetan, Paul; Zhang, Bai; Chen, Lily; Zhang, Zhen; Zhang, Hui

    2014-04-28

    Proteome Discoverer is one of many tools used for protein database search and peptide to spectrum assignment in mass spectrometry-based proteomics. However, the inadequacy of conversion tools makes it challenging to compare and integrate its results to those of other analytical tools. Here we present M2Lite, an open-source, light-weight, easily pluggable and fast conversion tool. M2Lite converts proteome discoverer derived MSF files to the proteomics community defined standard - the mzIdentML file format. M2Lite's source code is available as open-source at https://bitbucket.org/paiyetan/m2lite/src and its compiled binaries and documentation can be freely downloaded at https://bitbucket.org/paiyetan/m2lite/downloads.

  2. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    Science.gov (United States)

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

  3. Dual role of YM1+ M2 macrophages in allergic lung inflammation

    NARCIS (Netherlands)

    Draijer, Christina; Robbe, Patricia; Boorsma, Carian E; Hylkema, Machteld N; Melgert, Barbro N

    2018-01-01

    Alternatively activated (M2 or YM1+) macrophages have been associated with the development of asthma but their contribution to disease initiation and progression remains unclear. To assess the therapeutic potential of modulating these M2 macrophages, we have studied inhibition of M2 polarisation

  4. Machine-to-Machine networks: integration of M2M networks into companies' administrative networks

    OpenAIRE

    Pointereau, Romain

    2013-01-01

    This analysis will address the technical, economic and regulatory aspects and will identify the position taken by the various market actors. Integration of M2M Networks into Companies' Administrative Networks. Integración de redes M2M en redes administrativas de las empresas. Integració de xarxes M2M en xarxes administratives de les empreses.

  5. Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

    Directory of Open Access Journals (Sweden)

    Christine Pampeno

    Full Text Available The laminin receptor (LamR is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C and PrP(Sc. Indeed, LamR is a receptor for PrP(C. Whether LamR interacts with PrP(Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

  6. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    KAUST Repository

    Fiorillo, Annarita

    2014-03-21

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  7. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    Directory of Open Access Journals (Sweden)

    Annarita Fiorillo

    Full Text Available The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3, unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  8. Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

    Science.gov (United States)

    Salari, Farhad; Vahedi, Fatemeh; Chamani, Jamshidkhan; Varasteh, Abdolreza; Ketabdar, Hanieh; Sankian, Mojtaba

    2015-01-01

    Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  9. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    KAUST Repository

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  10. The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

    Science.gov (United States)

    López-Carrasco, Amparo; Flores, Ricardo

    2017-07-01

    Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

  11. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms

    Science.gov (United States)

    Rőszer, Tamás

    2015-01-01

    The alternatively activated or M2 macrophages are immune cells with high phenotypic heterogeneity and are governing functions at the interface of immunity, tissue homeostasis, metabolism, and endocrine signaling. Today the M2 macrophages are identified based on the expression pattern of a set of M2 markers. These markers are transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. This review discusses whether these M2 markers can be reliably used to identify M2 macrophages and define their functional subdivisions. Also, it provides an update on the novel signals of the tissue environment and the neuroendocrine system which shape the M2 activation. The possible evolutionary roots of the M2 macrophage functions are also discussed. PMID:26089604

  12. M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A.

    Directory of Open Access Journals (Sweden)

    Lorena Itatí Ibañez

    Full Text Available The ectodomain of influenza A matrix protein 2 (M2e is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs. We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4(+ T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2 or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

  13. Collection and sharing of medical information and medical intelligence (M2I) in NATO: a transversal survey.

    Science.gov (United States)

    Bedubourg, Gabriel; Wiik, H; Queyriaux, B; Lausund, P; Meynard, J-B

    2018-04-06

    There is a requirement for adequate medical expertise to be available throughout the range of potential future operations involving members of the North Atlantic Treaty Organization (NATO). The aim of this study was to assess the collection and sharing of medical intelligence and medical information (M2I) by NATO Nations, Partner Nations and NATO Command Structure and NATO Force Structure Headquarters (NCS/NFS HQs). A transversal survey was conducted between December 2014 and March 2015 using a survey form on M2I sent to NATO Nations and Partnership for Peace (PfP) Nations as well as NCS/NFS HQs. Correctly completed responses were received from 15/40 (37.5%) of the possible NATO and PfP Nations (37.5%) and 7/8 (87.5%) of the NCS/NFS HQs (100.0%). Deficiencies in the collection of M2I data were found due to lack of specific doctrines, networks, tools, structures and organisation. The survey provided an indication even though the participation rate was low for Nations. Part of the problem is thought to be that medical information and medical intelligence often lie in different chains of command. Future directions for this research could include studying the possibilities of a new specific information technology (IT) system to collect and to share M2I. Collection and sharing of M2I within the NATO/PfP community requires facilitation in order to strengthen the basis for decision-making and force health protection. The development of a dedicated NATO IT system may be a precondition for the implementation of an efficient M2I network. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  14. Reexamination of M2,3 atomic level widths and L1M2,3 transition energies of elements 69≤Z≤95

    Science.gov (United States)

    Fennane, K.; Berset, M.; Dousse, J.-Cl.; Hoszowska, J.; Raboud, P.-A.; Campbell, J. L.

    2013-11-01

    We report on high-resolution measurements of the photoinduced L1M2 and L1M3 x-ray emission lines of 69Tm, 70Yb, 71Lu, 73Ta, 74W, 75Re, 77Ir, 81Tl, 83Bi, and 95Am. From the linewidths of the measured transitions an accurate set of M2 and M3 level widths is determined assuming for the L1 level widths the values reported by Raboud [P.-A. Raboud et al., Phys. Rev. APLRAAN1050-294710.1103/PhysRevA.65.022512 65, 022512 (2002)]. Furthermore, the present experimental M2,3 data set is extended to 80Hg, 90Th, and 92U, using former L1M2,3 high-resolution x-ray emission spectroscopy measurements performed by our group. A detailed comparison of the M2 and M3 level widths determined in the present work with those recommended by Campbell and Papp [J. L. Campbell and T. Papp, At. Data Nucl. Data TablesADNDAT0092-640X10.1006/adnd.2000.0848 77, 1 (2001)] and other available experimental data as well as theoretical predictions is done. The observed abrupt changes of the M2,3 level widths versus atomic number Z can be explained satisfactorily by the cutoffs and onsets of the M2M4N1, respectively M3M4N3,4,5 and M3M5N2,3 Coster-Kronig transitions deduced from the semiempirical (Z+1) approximation. As a spin-off result of this study, precise L1M2 and L1M3 transition energies are obtained for the investigated elements. A very good agreement with transition energies calculated within the many-body perturbation theory is found.

  15. Teaching Form as Form

    DEFF Research Database (Denmark)

    Keiding, Tina Bering

    2012-01-01

    understanding of form per se, or, to use an expression from this text, of form as form. This challenge can be reduced to one question: how can design teaching support students in achieving not only the ability to recognize and describe different form-related concepts in existing design (i.e. analytical...

  16. Inhibition of the pore-forming protein perforin by a series of aryl-substituted isobenzofuran-1(3H)-ones.

    Science.gov (United States)

    Spicer, Julie A; Huttunen, Kristiina M; Miller, Christian K; Denny, William A; Ciccone, Annette; Browne, Kylie A; Trapani, Joseph A

    2012-02-01

    An aryl-substituted isobenzofuran-1(3H)-one lead compound was identified from a high throughput screen designed to find inhibitors of the lymphocyte pore-forming protein perforin. A series of analogs were then designed and prepared, exploring structure-activity relationships through variation of 2-thioxoimidazolidin-4-one and furan subunits on an isobenzofuranone core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 natural killer effector cells was determined. Several compounds showed excellent activity at concentrations that were non-toxic to the killer cells. This series represents a significant improvement on previous classes of compounds, being substantially more potent and largely retaining activity in the presence of serum. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

    Science.gov (United States)

    Awayn, N H; Rosenberg, M F; Kamis, A B; Aleksandrov, L A; Riordan, J R; Ford, R C

    2005-11-01

    Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

  18. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

    Science.gov (United States)

    Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

    2016-10-01

    The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

  19. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

    Directory of Open Access Journals (Sweden)

    Sébastien Besteiro

    2009-02-01

    Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

  20. Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

    Science.gov (United States)

    Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

    1996-01-01

    To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

  1. Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

    Science.gov (United States)

    Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

    2012-02-10

    The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

  2. Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

    Science.gov (United States)

    Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

    2012-01-01

    The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

  3. Disruption of the M2 gene of murine gammaherpesvirus 68 alters splenic latency following intranasal, but not intraperitoneal, inoculation.

    Science.gov (United States)

    Jacoby, Meagan A; Virgin, Herbert W; Speck, Samuel H

    2002-02-01

    Infection of mice with murine gammaherpesvirus 68 (gamma HV68; also referred to as MHV68) provides a tractable small-animal model with which to address the requirements for the establishment and maintenance of gammaherpesvirus infection in vivo. The M2 gene of gamma HV68 is a latency-associated gene that encodes a protein lacking discernible homology to any known viral or cellular proteins. M2 gene transcripts have been detected in latently infected splenocytes (S. M. Husain, E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, and J. T. Sample, Proc. Natl. Acad. Sci. USA 96:7508-7513, 1999; H. W. Virgin IV, R. M. Presti, X. Y. Li, C. Liu, and S. H. Speck, J. Virol. 73:2321-2332, 1999) and peritoneal exudate cells (H. W. Virgin IV, R. M. Presti, X. Y. Li, C. Liu, and S. H. Speck, J. Virol. 73:2321-2332, 1999), as well as in a latently gamma HV68-infected B-lymphoma cell line (S. M. Husain, E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, and J. T. Sample, Proc. Natl. Acad. Sci. USA 96:7508-7513, 1999). Here we describe the generation of gamma HV68 mutants with disruptions in the M2 gene. Mutation of the M2 gene did not affect the ability of the virus to replicate in tissue culture, nor did it affect gamma HV68 virulence in B6.Rag1 deficient mice. However, we found that M2 was differentially required for acute replication in vivo. While mutation of M2 did not affect acute phase of virus replication in the lungs of mice following intranasal inoculation, acute-phase virus replication in the spleen was decreased compared to that of the wild-type and marker rescue viruses following intraperitoneal inoculation. Upon intranasal inoculation, M2 mutant viruses exhibited a significant decrease in the establishment of latency in the spleen on day 16 postinfection, as measured by the frequency of viral genome-positive cells. In addition, M2 mutant viral genome

  4. The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

    Science.gov (United States)

    Xue, Yutong; Gibbons, Richard; Yan, Zhijiang; Yang, Dafeng; McDowell, Tarra L; Sechi, Salvatore; Qin, Jun; Zhou, Sharleen; Higgs, Doug; Wang, Weidong

    2003-09-16

    ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

  5. Detection of AA76, a Common Form of Amyloid A Protein, as a Way of Diagnosing AA Amyloidosis.

    Science.gov (United States)

    Sato, Junji; Okuda, Yasuaki; Kuroda, Takeshi; Yamada, Toshiyuki

    2016-01-01

    Reactive amyloid deposits consist of amyloid A (AA) proteins, the degradation products of serum amyloid A (SAA). Since the most common species of AA is the amino terminal portion produced by cleavage between residues 76 and 77 of SAA (AA76), the presence of AA76 in tissues could be a consequence of AA amyloid deposition. This study assessed the diagnostic significance of the detection of AA76 for AA amyloidosis using two different approaches. Biopsy specimens (n=130 from 54 subjects) from gastroduodenal mucosa or abdominal fat (n=9 from 9 subjects) of patients who had already been diagnosed with or were suspected of having AA amyloidosis were used. Fixed mucosal sections were subjected to immunohistochemistry using a newly developed antibody recognizing the carboxyl terminal end of AA76 (anti-AA76). The non-fixed materials from gastroduodenal mucosa or abdominal fat were subjected to immunoblotting for detection of the size of AA76. Among the gastroduodenal specimens (n=115) from already diagnosed patients, the positive rates of Congo red staining, immunohistochemistry using anti-AA76, and immunoblotting were 68.4%, 73.0%, and 92.2%, respectively. The anti-AA76 did not stain the supposed SAA in the blood or leakage, which was stained by anti-SAA antibody. AA76 was not detected either by immunohistochemistry or by immunoblot in the materials from patients in whom AA amyloidosis had been ruled out. In the abdominal fat, the immunoblot detected AA76 in 8 materials from 8 already diagnosed patients and did not in 1 patient whose gastroduodenal mucosa was negative. In conclusion, the detection of AA76 may alter the ability to diagnose AA amyloidosis. In immunohistochemistry for fixed specimens, the new anti-AA76 antibody can improve the specificity. Immunoblot for non-fixed materials, which can considerably improve the sensitivity, should be beneficial for small materials like abdominal fat. © 2016 by the Association of Clinical Scientists, Inc.

  6. An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.

    Science.gov (United States)

    Alexandrescu, A T

    2001-01-01

    Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (approximately 1 x 10(-3) min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10(-5) min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are approximately 10(3), and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10(5) on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

  7. β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Science.gov (United States)

    Lamkin, Donald M.; Ho, Hsin-Yun; Ong, Tiffany H.; Kawanishi, Carly K.; Stoffers, Victoria L.; Ahlawat, Nivedita; Ma, Jeffrey C.Y.; Arevalo, Jesusa M. G.; Cole, Steve W.; Sloan, Erica K.

    2016-01-01

    β-adrenergic signaling can regulate macrophage involvement in several diseases and often produces anti-inflammatory properties in macrophages, which are similar to M2 properties in a dichotomous M1 vs. M2 macrophage taxonomy. However, it is not clear that β-adrenergic-stimulated macrophages may be classified strictly as M2. In this in vitro study, we utilized recently published criteria and transcriptome-wide bioinformatics methods to map the relative polarity of murine β-adrenergic-stimulated macrophages within a wider M1–M2 spectrum. Results show that β-adrenergic-stimulated macrophages did not fit entirely into any one predefined category of the M1–M2 spectrum but did express genes that are representative of some M2 side categories. Moreover, transcript origin analysis of genome-wide transcriptional profiles located β-adrenergic-stimulated macrophages firmly on the M2 side of the M1–M2 spectrum and found active suppression of M1 side gene transcripts. The signal transduction pathways involved were mapped through blocking experiments and bioinformatics analysis of transcription factor binding motifs. M2-promoting effects were mediated specifically through β2-adrenergic receptors and were associated with CREB, C/EBPβ, and ATF transcription factor pathways but not with established M1–M2 STAT pathways. Thus, β-adrenergic-signaling induces a macrophage transcriptome that locates on the M2 side of the M1–M2 spectrum but likely accomplishes this effect through a signaling pathway that is atypical for M2-spectrum macrophages. PMID:27485040

  8. Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

    Directory of Open Access Journals (Sweden)

    Pallavi A Bafna

    2008-06-01

    Full Text Available Gating of nicotinic acetylcholine receptors from a C(losed to an O(pen conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where CO gating motions generate a lowhigh change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'. This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268 appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.

  9. Dispersing Si{sub 3}N{sub 4} at high solids loading - applied to protein forming

    Energy Technology Data Exchange (ETDEWEB)

    Lyckfeldt, O.; Palmqvist, L. [Swedish Ceramic Inst., Goeteborg (Sweden); Poeydemenge, F. [ENSCI, Limoges (France)

    2002-07-01

    The dispersing of a Si{sub 3}N{sub 4} powder (UBE SN-E10) at high solids loading in aqueous media was investigated. The powder was used in the as-received (raw) state, after thermal (calcinations) and/or mechanical pre-treatments (ball milling{yields}freeze granulation{yields}freeze-drying). Slips were prepared using pH adjustment with NH{sub 4}OH or an addition of Tiron (low-M{sub w} sulphonic acid). Zeta potential measurements of diluted systems and rheological evaluations of concentrated suspensions were conducted. The effect of adding whey protein concentrate (WPC) was also studied. Zeta potential measurements showed a clear decrease in pH{sub iep} by calcination, whereas Tiron slightly increased the pH{sub iep} of calcined powder and decreased the pH{sub iep} of the as-received powder. Rheological data showed that pH adjustment to 10 was more efficient in stabilising the as-received powder than the calcined powder. pH adjustment was also considered to be the most important effect of adding small amounts of Tiron (0.08 wt%). However, for calcined powder, Tiron was shown to be equally efficient as pH adjustment. Pre-milling followed by freeze granulation/freeze-drying resulted in de-agglomerated powders with improved ability to rapidly disperse and, hence, extend the possibility of achieving extreme solids loadings. When approaching the practical limits in solids loading of these pre-milled powders, slips with 49.5 vol% of as-received and 46.6 vol% of calcined powders displayed clear shear thickening behaviour. However, addition of WPC (12 wt% based on water) significantly decreased the degree of shear thickening although the viscosity at lower shear rates increased. The gelling of WPC was distinct and rapid in suspensions with the two pre-milled powders, as-received stabilised at pH 10 and calcined stabilised with Tiron. (orig.)

  10. Theoretical descriptions of novel triplet germylenes M1-Ge-M2-M3 (M1 = H, Li, Na, K; M2 = Be, Mg, Ca; M3 = H, F, Cl, Br).

    Science.gov (United States)

    Kassaee, Mohamad Zaman; Ashenagar, Samaneh

    2018-02-06

    In a quest to identify new ground-state triplet germylenes, the stabilities (singlet-triplet energy differences, ΔE S-T ) of 96 singlet (s) and triplet (t) M 1 -Ge-M 2 -M 3 species were compared and contrasted at the B3LYP/6-311++G**, QCISD(T)/6-311++G**, and CCSD(T)/6-311++G** levels of theory (M 1  = H, Li, Na, K; M 2  = Be, Mg, Ca; M 3  = H, F, Cl, Br). Interestingly, F-substituent triplet germylenes (M 3  = F) appear to be more stable and linear than the corresponding Cl- or Br-substituent triplet germylenes (M 3  = Cl or Br). Triplets with M 1  = K (i.e., the K-Ge-M 2 -M 3 series) seem to be more stable than the corresponding triplets with M 1  = H, Li, or Na. This can be attributed to the higher electropositivity of potassium. Triplet species with M 3  = Cl behave similarly to those with M 3  = Br. Conversely, triplets with M 3  = H show similar stabilities and linearities to those with M 3  = F. Singlet species of formulae K-Ge-Ca-Cl and K-Ge-Ca-Br form unexpected cyclic structures. Finally, the triplet germylenes M 1 -Ge-M 2 -M 3 become more stable as the electropositivities of the α-substituents (M 1 and M 2 ) and the electronegativity of the β-substituent (M 3 ) increase.

  11. Possible Inhibitor from Traditional Chinese Medicine for the β Form of Calcium-Dependent Protein Kinase Type II in the Treatment of Major Depressive Disorder

    Directory of Open Access Journals (Sweden)

    Tzu-Chieh Hung

    2014-01-01

    Full Text Available Recently, an important topic of major depressive disorder (MDD had been published in 2013. MDD is one of the most prevalent and disabling mental disorders. Consequently, much research is being undertaken into the causes and treatment. It has been found that inhibition of the β form of calcium/calmodulin-dependent protein kinase type II (β-CaMKII can ameliorate the disorder. Upon screening the traditional Chinese medicine (TCM database by molecular docking, sengesterone, labiatic acid, and methyl 3-O-feruloylquinate were selected for molecular dynamics. After 20 ns simulation, the RMSD, total energy, and structure variation could define the protein-ligand interaction. Furthermore, sengesterone, the principle candidate compound, has been found to have an effect on the regulation of emotions and memory development. In structure variation, we find the sample functional group of important amino acids make the protein stable and have limited variation. Due to similarity of structure variations, we suggest that these compounds may have an effect on β-CaMKII and that sengesterone may have a similar efficacy as the control. However labiatic acid may be a stronger inhibitor of β-CaMKII based on the larger RMSD and variation.

  12. DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

    Directory of Open Access Journals (Sweden)

    Marcus Thein

    Full Text Available Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

  13. Distributed Energy-Efficient Topology Control Algorithm in Home M2M Networks

    OpenAIRE

    Lee, Chao-Yang; Yang, Chu-Sing

    2012-01-01

    Because machine-to-machine (M2M) technology enables machines to communicate with each other without human intervention, it could play a big role in sensor network systems. Through wireless sensor network (WSN) gateways, various information can be collected by sensors for M2M systems. For home M2M networks, this study proposes a distributed energy-efficient topology control algorithm for both topology construction and topology maintenance. Topology control is an effective method of enhancing e...

  14. A novel subnucleocapsid nanoplatform for mucosal vaccination against influenza virus that targets the ectodomain of matrix protein 2.

    Science.gov (United States)

    Hervé, Pierre-Louis; Raliou, Mariam; Bourdieu, Christiane; Dubuquoy, Catherine; Petit-Camurdan, Agnès; Bertho, Nicolas; Eléouët, Jean-François; Chevalier, Christophe; Riffault, Sabine

    2014-01-01

    In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

  15. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  16. Reliable Reporting for Massive M2M Communications with Periodic Resource Pooling

    DEFF Research Database (Denmark)

    Madueño, Germán Corrales; Stefanovic, Cedomir; Popovski, Petar

    2014-01-01

    This letter considers a wireless M2M communication scenario with a massive number of M2M devices. Each device needs to send its reports within a given deadline and with certain reliability, e.g., 99.99%. A pool of resources available to all M2M devices is periodically available for transmission...... to guarantee the desired reliability of the report delivery within the deadline. The fact that the pool of resources is used by a massive number of devices allows to base the dimensioning on the central limit theorem. The results are interpreted in the context of LTE, but they are applicable to any M2M...

  17. Electric conductivity of double fluorides in the systems M1F-Th(U)F4(M1=K, Tl) and M2F2-ThF4(M2=Ca, Sr, Ba)

    International Nuclear Information System (INIS)

    Murin, I.V.; Andreev, A.M.; Amelin, Yu.V.

    1982-01-01

    The temperature dependence of electric conductivity of some double fluorides formed in the systems M 1 F-Th(U)F 4 (M 1 =K, Tl) and M 2 F 2 -ThF 4 (M 2 =Ca, Sr, Ba) as well as UF 3 in a wide temperature range is studied. It is shown that the values of electric conductivity and activation energy of these fluorides depend on the compound structure and cation nature. The temperature electric conductivity dependence for double fluorides with the tysonite structure is close to the lanthanum fluoride dependence. Taking into account low electron electric conductivity component the conclusion is drawn that the investigated compounds can be used as solid electrolytes

  18. Seed coat color, weight and eye pattern inheritance in gamma-rays induced cowpea M2-mutant line

    Directory of Open Access Journals (Sweden)

    Reda M. Gaafar

    2016-06-01

    Full Text Available Gamma radiation is a very effective tool for inducing genetic variation in characters of many plants. Black seeds of M2 mutant were obtained after exposure of an Egyptian cowpea cultivar (Kaha 1 to a low dose of gamma rays. Segregation of seed coat color, weight of 100 seeds and seed eye pattern of the black seeds of this mutant line were further examined in this study. Four colors were observed for seed coat in the M3 plants ranging from cream to reddish brown and three eye patterns were distinguished from each other. SDS–PAGE of the seed storage proteins showed 18 protein bands; five of these bands disappeared in the seeds of M3 plants compared to M2 and M0 controls while other 5 protein bands were specifically observed in seeds of M3 plants. PCR analysis using twelve ISSR primers showed 47 polymorphic and 8 unique amplicons. The eight unique amplicons were characteristic of the cream coat color and brown wide eye pattern (M03-G10 while the polymorphic bands were shared by 6 coat-color groups. A PCR fragment of 850 bp was amplified, using primer HB-12, in M3-G04 which showed high-100 seed weight. These results demonstrated the mutagenic effects of gamma rays on seed coat color, weight of 100 seeds and eye pattern of cowpea M3 mutant plants.

  19. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

    Science.gov (United States)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

    2012-05-01

    Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

  20. Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

    Directory of Open Access Journals (Sweden)

    Clement A Meseda

    Full Text Available Antibodies to both infectious forms of vaccinia virus, the mature virion (MV and the enveloped virion (EV, as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

  1. Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

    Science.gov (United States)

    Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

    2017-06-21

    Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

  2. Protective role of NKT cells and macrophage M2-driven phenotype in bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Grabarz, Felipe; Aguiar, Cristhiane Favero; Correa-Costa, Matheus; Braga, Tárcio Teodoro; Hyane, Meire I; Andrade-Oliveira, Vinícius; Landgraf, Maristella Almeida; Câmara, Niels Olsen Saraiva

    2018-04-01

    Pulmonary fibrosis is a result of an abnormal wound healing in lung tissue triggered by an excessive accumulation of extracellular matrix proteins, loss of tissue elasticity, and debit of ventilatory function. NKT cells are a major source of Th1 and Th2 cytokines and may be crucial in the polarization of M1/M2 macrophages in pulmonary fibrogenesis. Although there appears to be constant scientific progress in that field, pulmonary fibrosis still exhibits no current cure. From these facts, we hypothesized that NKT cells could influence the development of pulmonary fibrosis via modulation of macrophage activation. Wild type (WT) and NKT type I cell-deficient mice (Jα18 -/- ) were subjected to the protocol of bleomycin-induced pulmonary fibrosis with or without treatment with NKT cell agonists α-galactosylceramide and sulfatide. The participation of different cell populations, collagen deposition, and protein levels of different cytokines involved in inflammation and fibrosis was evaluated. The results indicate a benign role of NKT cells in Jα18 -/- mice and in wild-type α-galactosylceramide-sulfatide-treated groups. These animals presented lower levels of collagen deposition, fibrogenic molecules such as TGF-β and vimentin and improved survival rates. In contrast, WT mice developed a Th2-driven response augmenting IL-4, 5, and 13 protein synthesis and increased collagen deposition. Furthermore, the arginase-1 metabolic pathway was downregulated in wild-type NKT-activated and knockout mice indicating lower activity of M2 macrophages in lung tissue. Hence, our data suggest that NKT cells play a protective role in this experimental model by down modulating the Th2 milieu, inhibiting M2 polarization and finally preventing fibrosis.

  3. Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein from Helicobacter pylori: Role of Multiple Metal Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rong; Munger, Christine; Asinas, Abdalin; Benoit, Stephane L.; Miller, Erica; Matte, Allan; Maier, Robert J.; Cygler, Miroslaw (McGill); (Georgia); (Biotech Res.)

    2010-10-22

    The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 {angstrom} resolution, bound to Cu{sup 2+} at 2.7 {angstrom} resolution, and bound to Ni{sup 2+} at 3.1 {angstrom} resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding. The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni{sup 2+} for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni{sup 2+}-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.

  4. Properties of M1-M2-Si-Al-O-N glasses (M1 = La or Nd, M2 = Y or Er)

    Energy Technology Data Exchange (ETDEWEB)

    Pomeroy, M.J.; Nestor, E.; Hampshire, S. [Limerick Univ. (Ireland). Materials and Surface Science Inst.; Ramesh, R. [Littelfuse Ireland, Dundalk, Co. Louth (Ireland)

    2002-07-01

    Mixed lanthanide cation oxynitride glasses have been prepared in the M1 - M2 - Si-Al-O-N systems where M1 = La or Nd and M2 = Y or Er. The densities ({rho}), Young's moduli (E), microhardnesses (H{sub v}), glass transition temperatures (T{sub g}), dilatometric softening temperatures (T{sub dil}) and coefficients of thermal expansion (CTE) of 13 glasses were determined. The molar volume values (MV) calculated from density data, E, H{sub v}, T{sub g}, T{sub dil} and CTE values were all found to vary linearly with the effective cation field strength arising from the M1 and M2 modifier cations. Least squares intercept and slope values are presented which correlate each property to effective cation field strength together with error values which arise from glass and specimen preparation and measurement inconsistencies. These linear correlations clearly indicate that the overall glass structure remains the same for each of the thirteen glasses with only the modifier cation(s) having any influence. This influence appears to be a cross-linking effect, the strength of which increases as the effective cation field strength of the M1, M2 modifiers increases. (orig.)

  5. Models for financial crisis detection in Indonesia based on M1, M2 per foreign exchange reverse, and M2 multiplier indicators

    Science.gov (United States)

    Sugiyanto; Zukhronah, Etik; Pratiwi, Esteti Sophia

    2017-12-01

    Indonesia has been hit by financial crisis in the middle of 1997. The financial crisis that has occurred gives a severe impact to the economy of Indonesia resulting the needs for a detection system of financial crisis. Crisis can be detected based on several indicators such as M1, M2 per foreign exchange reserves, and M2 multiplier. These three indicators can affect the exchange rate stability and may further affect the financial stability so that it can be one of the causes of the financial crisis. This research aims to determine the appropriate model that can detect the financial crisis in Indonesia. Markov switching is an alternative model that can be approach and used often for detecting financial crisis. We can determine the combination of volatility and Markov switching model with AR and volatility model are determined first. The results of this research are that M1 can be modelled by SWARCH (3, 1) while M2 per foreign research exchange reserves and M2 multiplier can be modelled by SWARCH(3,2).

  6. 12 CFR Appendix M2 to Part 226 - Actual Repayment Disclosures

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Actual Repayment Disclosures M2 Appendix M2 to Part 226 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE... nearest whole year if the estimate contains a fractional year less than 0.5, and rounded up to the nearest...

  7. Establishing a Research Center: The Minority Male Community College Collaborative (M2C3)

    Science.gov (United States)

    Wood, J. Luke; Urias, Marissa Vasquez; Harris, Frank, III

    2016-01-01

    This chapter describes the establishment of the Minority Male Community College Collaborative (M2C3), a research and practice center at San Diego State University. M2C3 partners with community colleges across the United States to enhance access, achievement, and success among men of color. This chapter begins with a description of the national…

  8. M2- and M5-branes in E11 current algebra formulation of M-theory

    Science.gov (United States)

    Shiba, Shotaro; Sugawara, Hirotaka

    2018-03-01

    Equations of motion for M2- and M5-branes are written down in the E11 current algebra formulation of M-theory. These branes correspond to currents of the second and the fifth rank antisymmetric tensors in the E11 representation, whereas the electric and magnetic fields (coupled to M2- and M5-branes) correspond to currents of the third and the sixth rank antisymmetric tensors, respectively. We show that these equations of motion have solutions in terms of the coordinates on M2- and M5-branes. We also discuss the geometric equations, and show that there are static solutions when M2- or M5-brane exists alone and also when M5-brane wraps around M2-brane. This situation is realized because our Einstein-like equation contains an extra term which can be interpreted as gravitational energy contributing to the curvature, thus avoiding the usual intersection rule.

  9. Reliable Radio Access for Massive Machine-to-Machine (M2M) Communication

    DEFF Research Database (Denmark)

    Madueño, Germán Corrales

    the service requirements can range from massive number of devices to ultra-reliable. This PhD thesis focuses on novel mechanisms to meet these requirements in a variety of wireless systems, from well-established technologies such as cellular networks, to emerging technologies like IEEE 802.11ah. Today...... an overwhelming 89% of the deployed M2M modules are GPRS-based. This motivates us to investigate the potential of GPRS as a dedicated M2M network. We show that by introducing minimal modifications to GPRS operation, a large number of devices can be reliably supported. Surprisingly, even though LTE is seen...... as the preferable solution for M2M, no mechanisms are in place to guarantee reliable M2M access. Contrary to mainstream solutions that focus on preventing overload, we introduce mechanisms to provide reliable M2M service. We also investigate what cellular networks can do about upcoming smart metering traffic...

  10. Evaluation of secure capability-based access control in the M2M local cloud platform

    DEFF Research Database (Denmark)

    Anggorojati, Bayu; Prasad, Neeli R.; Prasad, Ramjee

    2016-01-01

    delegation. Recently, the capability based access control has been considered as method to manage access in the Internet of Things (IoT) or M2M domain. In this paper, the implementation and evaluation of a proposed secure capability based access control in the M2M local cloud platform is presented......Managing access to and protecting resources is one of the important aspect in managing security, especially in a distributed computing system such as Machine-to-Machine (M2M). One such platform known as the M2M local cloud platform, referring to BETaaS architecture [1], which conceptually consists...... of multiple distributed M2M gateways, creating new challenges in the access control. Some existing access control systems lack in scalability and flexibility to manage access from users or entity that belong to different authorization domains, or fails to provide fine grained and flexible access right...

  11. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  12. An M2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent H5N1 influenza viruses

    Directory of Open Access Journals (Sweden)

    Chan Chris CS

    2010-01-01

    Full Text Available Abstract Background A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses. Results Our results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses. Conclusions These results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses.

  13. M2 Macrophages Play Critical Roles in Progression of Inflammatory Liver Disease in Hepatitis C Virus Transgenic Mice.

    Science.gov (United States)

    Ohtsuki, Takahiro; Kimura, Kiminori; Tokunaga, Yuko; Tsukiyama-Kohara, Kyoko; Tateno, Chise; Hayashi, Yukiko; Hishima, Tsunekazu; Kohara, Michinori

    2016-01-01

    Macrophages in liver tissue are widely defined as important inflammatory cells in chronic viral hepatitis due to their proinflammatory activity. We reported previously that interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) play significant roles in causing chronic hepatitis in hepatitis C virus (HCV) transgenic mice (S. Sekiguchi et al., PLoS One 7:e51656, 2012, http://dx.doi.org/10.1371/journal.pone.0051656). In addition, we showed that recombinant vaccinia viruses expressing an HCV nonstructural protein (rVV-N25) could protect against the progression of chronic hepatitis by suppression of macrophage activation. Here, we focus on the role of macrophages in liver disease progression in HCV transgenic mice and examine characteristic features of macrophages following rVV-N25 treatment. The number of CD11b(+) F4/80(+) CD11c(-) CD206(+) (M2) macrophages in the liver of HCV transgenic mice was notably increased compared to that of age-matched control mice. These M2 macrophages in the liver produced elevated levels of IL-6 and TNF-α. rVV-N25 infection suppressed the number and activation of M2 macrophages in liver tissue. These results suggested that inflammatory cytokines produced by M2-like macrophages contribute to the induction of chronic liver inflammation in HCV transgenic mice. Moreover, the therapeutic effect of rVV-N25 might be induced by the suppression of the number and activation of hepatic macrophages. HCV causes persistent infections that can lead to chronic liver diseases, liver fibrosis, and hepatocellular carcinoma; the search for an HCV curative is the focus of ongoing research. Recently, effective anti-HCV drugs have been developed; however, vaccine development still is required for the prevention and therapy of infection by this virus. We demonstrate here that M2 macrophages are important for the pathogenesis of HCV-caused liver diseases and additionally show that M2 macrophages contribute to the therapeutic mechanism observed following r

  14. M2-F1 lifting body and Paresev 1B on ramp

    Science.gov (United States)

    1963-01-01

    In this photo of the M2-F1 lifting body and the Paresev 1B on the ramp, the viewer sees two vehicles representing different approaches to building a research craft to simulate a spacecraft able to land on the ground instead of splashing down in the ocean as the Mercury capsules did. The M2-F1 was a lifting body, a shape able to re-enter from orbit and land. The Paresev (Paraglider Research Vehicle) used a Rogallo wing that could be (but never was) used to replace a conventional parachute for landing a capsule-type spacecraft, allowing it to make a controlled landing on the ground. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop

  15. PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.

    Science.gov (United States)

    Chevalier, Christophe; Al Bazzal, Ali; Vidic, Jasmina; Février, Vincent; Bourdieu, Christiane; Bouguyon, Edwige; Le Goffic, Ronan; Vautherot, Jean-François; Bernard, Julie; Moudjou, Mohammed; Noinville, Sylvie; Chich, Jean-François; Da Costa, Bruno; Rezaei, Human; Delmas, Bernard

    2010-04-23

    The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

  16. PB1-F2 Influenza A Virus Protein Adopts a β-Sheet Conformation and Forms Amyloid Fibers in Membrane Environments

    Science.gov (United States)

    Chevalier, Christophe; Al Bazzal, Ali; Vidic, Jasmina; Février, Vincent; Bourdieu, Christiane; Bouguyon, Edwige; Le Goffic, Ronan; Vautherot, Jean-François; Bernard, Julie; Moudjou, Mohammed; Noinville, Sylvie; Chich, Jean-François; Da Costa, Bruno; Rezaei, Human; Delmas, Bernard

    2010-01-01

    The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an α-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display β-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a β-sheet structure. Dynamic light scattering revealed that the presence of β-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation. PMID:20172856

  17. Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein.

    Science.gov (United States)

    Wilson, Rona; Plinston, Chris; Hunter, Nora; Casalone, Cristina; Corona, Cristiano; Tagliavini, Fabrizio; Suardi, Silvia; Ruggerone, Margherita; Moda, Fabio; Graziano, Silvia; Sbriccoli, Marco; Cardone, Franco; Pocchiari, Maurizio; Ingrosso, Loredana; Baron, Thierry; Richt, Juergen; Andreoletti, Olivier; Simmons, Marion; Lockey, Richard; Manson, Jean C; Barron, Rona M

    2012-07-01

    The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

  18. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

    Science.gov (United States)

    Li, Junjie; Xie, Jiangang; Liu, Shanshou; Li, Xiao; Zhang, Dongliang; Wang, Xianqi; Jiang, Jinquan; Hu, Wei; Zhang, Yuan; Jin, Boquan; Zhuang, Ran; Yin, Wen

    2018-04-25

    ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity. The RNA-RNP immunoprecipitation binding assay demonstrated a direct interaction between ADAR1 and miR-21 precursor. Significant up-regulation in IL-10 and down-regulation in miR-21 were observed in ADAR1-overexpressing macrophages. We evaluated miR-21 target mRNAs and macrophage polarization signaling pathways and found that forkhead box protein O1 (Foxo1) was up-regulated in cells that overexpressed ADAR1. In a mouse allogeneic skin transplantation model, grafts in the ADAR1-overexpressed group survived longer and suffered less immune cell infiltration. In ADAR1-overexpressed recipients, splenic macrophages were significantly polarized to M2, and levels of sera IL-10 were markedly higher than those in the control group. In summary, ADAR1 modulates macrophage M2 polarization via the ADAR1-miR-21-Foxo1-IL-10 axis, thereby suppressing allogeneic graft rejection.-Li, J., Xie, J., Liu, S., Li, X., Zhang, D., Wang, X., Jiang, J., Hu, W., Zhang, Y., Jin, B., Zhuang, R., Yin, W. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

  19. Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines.

    Science.gov (United States)

    Ghosh, Mousumi; Xu, Yong; Pearse, Damien D

    2016-01-13

    Microglia and macrophages play a central role in neuroinflammation. Pro-inflammatory cytokines trigger their conversion to a classically activated (M1) phenotype, sustaining inflammation and producing a cytotoxic environment. Conversely, anti-inflammatory cytokines polarize the cells towards an alternatively activated (M2), tissue reparative phenotype. Elucidation of the signal transduction pathways involved in M1 to M2 phenotypic conversion may provide insight into how the innate immune response can be harnessed during distinct phases of disease or injury to mediate neuroprotection and neurorepair. Microglial cells (cell line and primary) were subjected to combined cyclic adenosine monophosphate (cyclic AMP) and IL-4, or either alone, in the presence of pro-inflammatory mediators, lipopolysaccharide (LPS), or tumor necrosis factor-α (TNF-α). Their effects on the expression of characteristic markers for M1 and M2 microglia were assessed. Similarly, the M1 and M2 phenotypes of microglia and macrophages within the lesion site were then evaluated following a contusive spinal cord injury (SCI) to the thoracic (T8) spinal cord of rats and mice when the agents were administered systemically. It was demonstrated that cyclic AMP functions synergistically with IL-4 to promote M1 to M2 conversion of microglia in culture. The combination of cyclic AMP and IL-4, but neither alone, induced an Arg-1(+)/iNOS(-)cell phenotype with concomitant expression of other M2-specific markers including TG2 and RELM-α. M2-converted microglia showed ameliorated production of pro-inflammatory cytokines (TNF-α and IP-10) and reactive oxygen species, with no alteration in phagocytic properties. M2a conversion required protein kinase A (PKA), but not the exchange protein directly activated by cyclic AMP (EPAC). Systemic delivery of cyclic AMP and IL-4 after experimental SCI also promoted a significant M1 to M2a phenotypic change in microglia and macrophage population dynamics in the lesion

  20. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    DEFF Research Database (Denmark)

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  1. A pH-dependent conformational ensemble mediates proton transport through the influenza A/M2 protein†

    Science.gov (United States)

    Polishchuk, Alexei L.; Lear, James D.; Ma, Chunlong; Lamb, Robert A.; Pinto, Lawrence H.; DeGrado, William F.

    2010-01-01

    The influenza A M2 protein exhibits inwardly rectifying, pH-activated proton transport that saturates at low pH. A comparison of high-resolution structures of the transmembrane domain at high and low pH suggests that pH-dependent conformational changes may facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. Here, we show that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 pKas. We then develop and test two similar, quantitative mechanistic models of proton transport, where protonation shifts the equilibrium between structural states having different proton affinities and solvent accessibilities. The models account well for a collection of experimental data sets over a wide range of pHs and voltages and require only a small number of adjustable parameters to accurately describe the data. While the kinetic models do not require any specific conformation for the protein, they nevertheless are consistent with a large body of structural information based on high-resolution NMR and crystallographic structures, optical spectroscopy, and MD calculations. PMID:20968306

  2. He II Raman Scattered Line by Neutral Hydrogen in the Bipolar Platenary Nebula M2-9

    Directory of Open Access Journals (Sweden)

    Hee-Won Lee

    2001-06-01

    Full Text Available In the spectrum of the young bipolar planetary nebula M2-9 obtained from the 1.5 m telescope at the Cerro Tololo Inter-American Observatory, we detected the He~II feature at 6545 Å that are proposed to be formed via Raman scattering by atomic hydrogen. However, in the same spectrum, the He~II emission lines at 6527 Å and 6560 Å are absent, which implies that the He~II emission region is hidden from our line of sight and that the H~I scattering region is pretty much extended not to be obscured entirely. We performed photoionization computations to estimate the physical size of the He~II emission line region to be 1016 cm, from which the location and dimension of the obscuring circumstellar region are inferred and the temperature of the central star must exceed 105 K. The angular size of the circumstellar region responsible for the obscuration of the He~II emission region is ~ 1'' with the assumption of the distance 01 kpc to M2-9, which is consistent with the recent image of M2-9 obtained with the Hubble Space Telescope.

  3. Initial Search for Triggered Gamma Emission from {sup 178}Hf{sup m2} Using the YSU Miniball Array

    Energy Technology Data Exchange (ETDEWEB)

    Carroll, J. J.; Burnett, J.; Drummond, T.; Lepak, J.; Propri, R.; Smith, D. [Youngstown State University, Department of Physics and Astronomy, Center for Photon-Induced Processes (United States); Karamian, S. A.; Adam, J. [Joint Institute for Nuclear Research (Russian Federation); Stedile, F. [Universitaet Stuttgart, Institut fuer Strahlenphysik (Germany); Agee, F. J. [Air Force Office of Scientific Research, AFOSR/NE (United States)

    2002-11-15

    Experiments with the long-lived, high-K isomer {sup 178}Hf{sup m2} have been recognized as intriguing tests of multi-quasiparticle state structures and their interactions with external radiation. A triggered release of the energy stored by this isomer, 2.5 MeV per nucleus or 1.2 GJ/gram, in the form of a gamma-ray burst might prove valuable for numerous applications. The observation of 'accelerated' decay of {sup 178}Hf{sup m2} during irradiation by 90-keV bremsstrahlung has already been reported, but with poor statistical accuracy due to the experimental approach. That approach employed single Ge detectors to seek increases in the areas of peaks at energies corresponding to transitions in the spontaneous decay of the isomer. The need for better quality data to confirm those results has motivated the development of improved detection concepts. One such concept was utilized here to perform an initial search for low-energy (<20 keV) triggered gamma emission from {sup 178}Hf{sup m2} using the YSU miniball detector array.

  4. Beta3 adrenoceptors substitute the role of M(2) muscarinic receptor in coping with cold stress in the heart: evidence from M(2)KO mice.

    Science.gov (United States)

    Benes, Jan; Novakova, Martina; Rotkova, Jana; Farar, Vladimir; Kvetnansky, Richard; Riljak, Vladimir; Myslivecek, Jaromir

    2012-07-01

    We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

  5. Nanosecond dynamics of influenza A/M2TM and an amantadine resistant mutant probed by time-dependent red shifts of a native tryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Nanda, Vikas [Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School – UMDNJ, Piscataway, NJ 08854 (United States); Department of Biochemistry, Robert Wood Johnson Medical School – UMDNJ, Piscataway, NJ 08854 (United States); Cristian, Lidia [Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059 (United States); Toptygin, Dmitri; Brand, Ludwig [Department of Biology, Johns Hopkins University, Baltimore, MD 21218 (United States); DeGrado, William F., E-mail: William.Degrado@ucsf.edu [Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158 (United States)

    2013-08-30

    Highlights: ► Examined nanosecond dynamics of essential tryptophan residue of M2 proton channel. ► Channel blocking drugs restrict the ability of M2 to stabilize charge. ► Dielectric relaxation of M2 consistent with molecular dynamics simulation studies. - Abstract: Proteins involved in functions such as electron transfer or ion transport must be capable of stabilizing transient charged species on time scales ranging from picoseconds to microseconds. We study the influenza A M2 proton channel, containing a tryptophan residue that serves as an essential part of the proton conduction pathway. We induce a transition dipole in tryptophan by photoexcitation, and then probe the dielectric stabilization of its excited state. The magnitude of the stabilization over this time regime was larger than that generally found for tryptophan in membrane or protein environments. M2 achieves a water-like stabilization over a 25 ns time scale, slower than that of bulk water, but sufficiently rapid to contribute to stabilization of charge as protons diffuse through the channel. These measurements should stimulate future MD studies to clarify the role of sidechain versus non-bulk water in defining the process of relaxation.

  6. Comparison of 14 MeV isomer production of 178m2Hf and 179m2Hf using Feshbach-Kerman-Koonin and exciton preequilibrium models

    International Nuclear Information System (INIS)

    Chadwick, M.B.; Young, P.G.

    1993-01-01

    The 178m2 Hf(16+) isomeric state has a 31-yr half life and could pose serious radioactive problems in nuclear fusion reactors if its production in 14 MeV neutron-induced reactions is significant. We present statistical/preequilibrium model calculations for the production of this isomer in the 179 Hf(n, 2n) 178m2 Hf reaction, as well as the 25-days 12.5 - isomer in the 179 Hf(n,n') 179m2 Hf reaction, using two different preequilibrium models: the exciton model and the Feshbach-Kerman-Koonin (FKK) theory. Our calculations which use the exciton model agree well with measurements, but those with the FKK theory underestimate measurements. Our calculations axe the first to probe angular momentum transfer effects in the FKK theory and suggest that, as it is presently applied, high spin-transfer reactions are underestimated. We suggest modifications to the FKK statistical averaging procedure which may result in an improved agreement with experiment

  7. Point mutation of a conserved aspartate, D69, in the muscarinic M2 receptor does not modify voltage-sensitive agonist potency.

    Science.gov (United States)

    Ågren, Richard; Sahlholm, Kristoffer; Nilsson, Johanna; Århem, Peter

    2018-01-29

    The muscarinic M 2 receptor (M 2 R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M 2 R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M 2 R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC 50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M 2 R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M 2 R. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Ab initio study of M2AlN (M = Ti,V,Cr)

    International Nuclear Information System (INIS)

    Sun, Zhimei; Music, Denis; Ahuja, Rajeev; Schneider, Jochen M

    2005-01-01

    We have studied M 2 AlN phases, where M = Ti, V, and Cr, by means of ab initio total energy calculations. The bulk modulus of M 2 AlN increases as Ti is replaced with V and Cr by 19.0% and 26.5%, respectively, which can be understood on the basis of the increased number of valence electrons filling the p-d hybridized bonding states. The bulk modulus of M 2 AlN is generally higher than that of the corresponding M 2 AlC phase, which may be explained by an extra electron in the former phases contributing to stronger chemical bonding. This work is important for fundamental understanding of elastic properties of these ternary nitrides and may inspire future experimental research. (letter to the editor)

  9. HF183/BFDrev and HumM2 qPCR data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Concentration estimates for HF183/BFDrev and HumM2 qPCR genetic markers in raw sewage collected from 54 geographic locations across the United States. This dataset...

  10. 10D massive type IIA supergravities as the uplift of parabolic M2-brane torus bundles

    Energy Technology Data Exchange (ETDEWEB)

    Garcia del Moral, Maria Pilar [Universidad de Antofagasta (Chile). Dept. de Fisica; Restuccia, Alvaro [Universidad de Antofagasta (Chile). Dept. de Fisica; Universidad Simon Bolivar, Caracas (Venezuela, Bolivarian Republic of). Dept. de Fisica

    2016-04-15

    We remark that the two 10D massive deformations of the N = 2 maximal type IIA supergravity (Romans and HLW supergravity) are associated to the low energy limit of the uplift to 10D of M2-brane torus bundles with parabolic monodromy linearly and non-linearly realized respectively. Romans supergravity corresponds to M2-brane compactified on a twice-punctured torus bundle. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  11. Enhanced M1/M2 macrophage ratio promotes orthodontic root resorption.

    Science.gov (United States)

    He, D; Kou, X; Luo, Q; Yang, R; Liu, D; Wang, X; Song, Y; Cao, H; Zeng, M; Gan, Y; Zhou, Y

    2015-01-01

    Mechanical force-induced orthodontic root resorption is a major clinical challenge in orthodontic treatment. Macrophages play an important role in orthodontic root resorption, but the underlying mechanism remains unclear. In this study, we examined the mechanism by which the ratio of M1 to M2 macrophage polarization affects root resorption during orthodontic tooth movement. Root resorption occurred when nickel-titanium coil springs were applied on the upper first molars of rats for 3 to 14 d. Positively stained odontoclasts or osteoclasts with tartrate-resistant acid phosphatase were found in resorption areas. Meanwhile, M1-like macrophages positive for CD68 and inducible nitric oxide synthase (iNOS) persistently accumulated on the compression side of periodontal tissues. In addition, the expressions of the M1 activator interferon-γ and the M1-associated pro-inflammatory cytokine tumor necrosis factor (TNF)-α were upregulated on the compression side of periodontal tissues. When the coil springs were removed at the 14th day after orthodontic force application, root resorption was partially rescued. The number of CD68(+)CD163(+) M2-like macrophages gradually increased on the compression side of periodontal tissues. The levels of M2 activator interleukin (IL)-4 and the M2-associated anti-inflammatory cytokine IL-10 also increased. Systemic injection of the TNF-α inhibitor etanercept or IL-4 attenuated the severity of root resorption and decreased the ratio of M1 to M2 macrophages. These data imply that the balance between M1 and M2 macrophages affects orthodontic root resorption. Root resorption was aggravated by an enhanced M1/M2 ratio but was partially rescued by a reduced M1/M2 ratio. © International & American Associations for Dental Research 2014.

  12. Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

    Science.gov (United States)

    Carlson, Signe; Helterline, Deri; Asbe, Laura; Dupras, Sarah; Minami, Elina; Farris, Stephen; Stempien-Otero, April

    2017-07-01

    Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, Padopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Detailed profile of m=2 islands with TVTS on JFT-2M

    International Nuclear Information System (INIS)

    Yamauchi, T.; Grek, B.; Hoshino, K.; Le Blanc, B.; Johnson, D.; Felt, J.; Shiina, T.; Kurita, G.; Ishige, Y.; Kozawa, H.

    1996-01-01

    The detailed electron temperature profile (spatial resolution: 0.86 cm) of a low density JFT-2M plasma is measured with the TV Thomson scattering system (TVTS). Flat profiles showing the electron temperature shapes of m=2/n=1 islands are presented, which are in contrast to that without islands. On the other hand, the m=2/n=1 islands are effectively suppressed with local ECRH heating. (orig.)

  14. Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

    Science.gov (United States)

    Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

    2017-10-01

    Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

  15. Cyclooxygenase-2 inhibition blocks M2 macrophage differentiation and suppresses metastasis in murine breast cancer model.

    Directory of Open Access Journals (Sweden)

    Yi-Rang Na

    Full Text Available Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2 inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFβ. In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis.

  16. The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

    Energy Technology Data Exchange (ETDEWEB)

    Makhov, A.M.; Simon, M.; Sen, A.; Yu, X.; Griffith, J. D.; Egelman, E. H.

    2009-02-20

    Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is {approx} 250 {angstrom}, with {approx} 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing {approx} 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

  17. The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

    Science.gov (United States)

    Nagata, Mariko; Ishino, Sonoko; Yamagami, Takeshi; Ogino, Hiromi; Simons, Jan-Robert; Kanai, Tamotsu; Atomi, Haruyuki; Ishino, Yoshizumi

    2017-10-13

    The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

  19. Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro.

    Science.gov (United States)

    Chien, H H; Lin, W L; Cho, M I

    1999-05-01

    The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture

  20. Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides.

    Science.gov (United States)

    Pannell, Maria; Labuz, Dominika; Celik, Melih Ö; Keye, Jacqueline; Batra, Arvind; Siegmund, Britta; Machelska, Halina

    2016-10-07

    During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 10 5 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and β-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the

  1. Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

    Energy Technology Data Exchange (ETDEWEB)

    Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Thulin, Petra; Ehrenborg, Ewa [Atherosclerosis Research Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm (Sweden); Olivecrona, Thomas [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Olivecrona, Gunilla, E-mail: Gunilla.Olivecrona@medbio.umu.se [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

  2. Radiographic and Histologic Evaluation of a Bone Void that Formed After Recombinant Human Bone Morphogenetic Protein-2-Mediated Sinus Graft Augmentation: A Case Report.

    Science.gov (United States)

    Kang, Hyun-Joo; Jun, Choong-Man; Yun, Jeong-Ho

    2016-01-01

    In the present case report, the authors describe radiographic and histologic observations of a bone void that formed after a sinus augmentation using a graft material that contained recombinant human bone morphogenetic protein-2 (rhBMP-2) and discuss clinical and histologic implications of their findings. Sinus augmentation was performed using a graft material comprising 1 g of hydroxyapatite/β-tricalcium phosphate, which contained 1 mg of rhBMP-2. Radiographic evaluation was conducted with panoramic radiographs and computed tomography images of the augmented maxillary sinus, which were analyzed using a three-dimensional image-reconstruction program. Histologic evaluation was also performed on a biopsy specimen obtained 6 months after the sinus augmentation. The total augmented volume increased from 1,582.2 mm(3) immediately after the sinus augmentation to 3,344.9 mm3 at 6 months after the augmentation because of the formation of a bone void. Twenty-six months after the sinus augmentation, the bone void remained but had reduced in volume, with the total augmented volume reduced to 2,551.7 mm(3). Histologically, new bone was observed to be in contact with the grafted particles, and a fatty marrow-like tissue was present in the area of the bone void. This case report shows that the bone void that had formed after sinus augmentation resolved over time and seemed to be partially replaced with new bone. Furthermore, none of the implants failed, and clinical adverse events were not observed during the follow-up period.

  3. Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

    International Nuclear Information System (INIS)

    Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

    2012-01-01

    Highlights: ► Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. ► Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. ► Only monomers of ANGPTL4 are present within THP-1 macrophages. ► Covalent oligomers of ANGPTL4 appear on cell surface and in medium. ► Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

  4. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-11-01

    Full Text Available VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa, and the shorter VirF21 (21 kDa, lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.

  5. {sup 13}C, {sup 15}N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

    Energy Technology Data Exchange (ETDEWEB)

    Siemer, Ansgar B. [Physical Chemistry (Switzerland); Ritter, Christiane [Salk Institute, Structural Biology Laboratory (United States); Steinmetz, Michel O. [Paul Scherrer Institut, Biomolecular Research, Structural Biology (Switzerland); Ernst, Matthias [Physical Chemistry (Switzerland); Riek, Roland [Salk Institute, Structural Biology Laboratory (United States); Meier, Beat H. [Physical Chemistry (Switzerland)

    2006-02-15

    The partial {sup 15}N and {sup 13}C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional {sup 13}C--{sup 13}C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional {sup 15}N--{sup 13}C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues.

  6. Bovine colostrum increases pore-forming claudin-2 protein expression but paradoxically not ion permeability possibly by a change of the intestinal cytokine milieu.

    Directory of Open Access Journals (Sweden)

    Peggy Bodammer

    Full Text Available An impaired intestinal barrier function is involved in the pathogenesis of inflammatory bowel disease (IBD. Several nutritional factors are supposed to be effective in IBD treatment but scientific data about the effects on the intestinal integrity remain scarce. Bovine colostrum was shown to exert beneficial effects in DSS-induced murine colitis, and the present study was undertaken to explore the underlying molecular mechanisms. Western blot revealed increased claudin-2 expression in the distal ileum of healthy mice after feeding with colostrum for 14 days, whereas other tight junction proteins (claudin-3, 4, 10, 15 remained unchanged. The colostrum-induced claudin-2 induction was confirmed in differentiated Caco-2 cells after culture with colostrum for 48 h. Paradoxically, the elevation of claudin-2, which forms a cation-selective pore, was neither accompanied by increased ion permeability nor impaired barrier function. In an in situ perfusion model, 1 h exposure of the colonic mucosa to colostrum induced significantly increased mRNA levels of barrier-strengthening cytokine transforming growth factor-β, while interleukine-2, interleukine-6, interleukine-10, interleukine-13, and tumor-necrosis factor-α remained unchanged. Thus, modulation of the intestinal transforming growth factor-β expression might have compensated the claudin-2 increase and contributed to the observed barrier strengthening effects of colostrum in vivo and in vitro.

  7. Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

    Science.gov (United States)

    Kim, MinSung; Jung, Won-Kyo; Kim, GeunHyung

    2013-11-01

    Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

  8. MD SIMULATION STUDIES TO INVESTIGATE ISO-ENERGETIC CONFORMATIONAL BEHAVIOUR OF MODIFIED NUCLEOSIDES M2G AND M22G PRESENT IN tRNA

    Directory of Open Access Journals (Sweden)

    Rohit S Bavi

    2013-02-01

    Full Text Available Modified nucleic acid bases are most commonly found in tRNA. These may contain modifications from simple methylation to addition of bulky groups. Methylation of the four canonical nucleotide bases at a wide variety of positions is particularly prominent among the known modification. Methylation of N2 group of guanine is a relatively common modification in tRNA and rRNA. N2-methylguanosine (m2G is the second most often encountered nucleoside in E. coli tRNAs. N2, N2-dimethylguanosine (m22G is found in the majority of eukaryotic tRNAs and involved in forming base pair interactions with adjacent bases. Hence, in order to understand the structural significance of these methylated nucleic acid bases we have carried out molecular dynamics simulation to see the salvation effect. The results obtained shows iso-energetic conformational behaviors for m2G and m22G. The simulation trajectory of m2G shows regular periodical fluctuations suggesting that m2G is equally stable as either s-cis or s-trans rotamers. The two rotamers of m2G may interact canonically or non-canonically with opposite base as s-trans m2G26:C/A/U44 and s-cis m2G26:A/U44. The free rotations around the C-N bond could be the possible reason for these iso-energetic conformations. Dimethylation of G has almost no influence on base pairing with either A or U. Thus, these results reveal that modified nucleosides m2G and m22G may play an important role to prevent tRNA from adopting the unusual mitochondrial like conformation.

  9. The challenges of M2M massive access in wireless cellular networks

    Directory of Open Access Journals (Sweden)

    Andrea Biral

    2015-02-01

    Full Text Available The next generation of communication systems, which is commonly referred to as 5G, is expected to support, besides the traditional voice and data services, new communication paradigms, such as Internet of Things (IoT and Machine-to-Machine (M2M services, which involve communication between Machine-Type Devices (MTDs in a fully automated fashion, thus, without or with minimal human intervention. Although the general requirements of 5G systems are progressively taking shape, the technological issues raised by such a vision are still partially unclear. Nonetheless, general consensus has been reached upon some specific challenges, such as the need for 5G wireless access networks to support massive access by MTDs, as a consequence of the proliferation of M2M services. In this paper, we describe the main challenges raised by the M2M vision, focusing in particular on the problems related to the support of massive MTD access in current cellular communication systems. Then we analyze the most common approaches proposed in the literature to enable the coexistence of conventional and M2M services in the current and next generation of cellular wireless systems. We finally conclude by pointing out the research challenges that require further investigation in order to provide full support to the M2M paradigm.

  10. High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.

    Science.gov (United States)

    Reddy, S G; Cochran, B J; Worth, L L; Knutson, V P; Haddox, M K

    1994-04-01

    A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

  11. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator.

    Science.gov (United States)

    Di Martino, Maria Letizia; Romilly, Cédric; Wagner, E Gerhart H; Colonna, Bianca; Prosseda, Gianni

    2016-11-08

    VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF 30 (30 kDa), and the shorter VirF 21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF 30 and VirF 21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF 21 is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF 21 The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF 30 is responsible for activation of the virulence system, VirF 21 negatively autoregulates virF expression itself. Since VirF 21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes

  12. Probing topology and dynamics of the second transmembrane domain (M2δ) of the acetyl choline receptor using magnetically aligned lipid bilayers (bicelles) and EPR spectroscopy.

    Science.gov (United States)

    Sahu, Indra D; Mayo, Daniel J; Subbaraman, Nidhi; Inbaraj, Johnson J; McCarrick, Robert M; Lorigan, Gary A

    2017-08-01

    Characterizing membrane protein structure and dynamics in the lipid bilayer membrane is very important but experimentally challenging. EPR spectroscopy offers a unique set of techniques to investigate a membrane protein structure, dynamics, topology, and distance constraints in lipid bilayers. Previously our lab demonstrated the use of magnetically aligned phospholipid bilayers (bicelles) for probing topology and dynamics of the membrane peptide M2δ of the acetyl choline receptor (AchR) as a proof of concept. In this study, magnetically aligned phospholipid bilayers and rigid spin labels were further utilized to provide improved dynamic information and topology of M2δ peptide. Seven TOAC-labeled AchR M2δ peptides were synthesized to demonstrate the utility of a multi-labeling amino acid substitution alignment strategy. Our data revealed the helical tilts to be 11°, 17°, 9°, 17°, 16°, 11°, 9°±4° for residues I7TOAC, Q13TOAC, A14TOAC, V15TOAC, C16TOAC, L17TOAC, and L18TOAC, respectively. The average helical tilt of the M2δ peptide was determined to be ∼13°. This study also revealed that the TOAC labels were attached to the M2δ peptide with different dynamics suggesting that the sites towards the C-terminal end are more rigid when compared to the sites towards the N-terminus. The dynamics of the TOAC labeled sites were more resolved in the aligned samples when compared to the randomly disordered samples. This study highlights the use of magnetically aligned lipid bilayer EPR technique to determine a more accurate helical tilt and more resolved local dynamics of AchR M2δ peptide. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Aggregation and Trunking of M2M Traffic via D2D Connections

    DEFF Research Database (Denmark)

    Rigazzi, Giovanni; Kiilerich Pratas, Nuno; Popovski, Petar

    2015-01-01

    Machine-to-Machine (M2M) communications is one of the key enablers of the Internet of Things (IoT). Billions of devices are expected to be deployed in the near future for novel M2M applications demanding ubiquitous access and global connectivity. In order to cope with the massive number of machines......, there is a need for new techniques to coordinate the access and allocate the resources. Although the majority of the proposed solutions are focused on the adaptation of the traditional cellular networks to the M2M traffic patterns, novel approaches based on the direct communication among nearby devices may...... represent an effective way to avoid access congestion and cell overload. In this paper, we propose a new strategy inspired by the classical Trunked Radio Systems (TRS), exploiting the Device-to-Device (D2D) connectivity between cellular users and Machine-Type Devices (MTDs). The aggregation of the locally...

  14. M2-F1 in flight being towed by a C-47

    Science.gov (United States)

    1964-01-01

    The M2-F1 Lifting Body is seen here being towed behind a C-47 at the Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. In this rear view, the M2-F1 is flying above and to one side of the C-47. This was done to avoid wake turbulence from the towplane. Lacking wings, the M2-F1 used an unusual configuration for its control surfaces. It had two rudders on the fins, two elevons (called 'elephant ears') mounted on the outsides of the fins, and two body flaps on the upper rear fuselage. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. These initial tests produced enough flight data about the M2-F1 to proceed with flights behind the C-47 tow plane at greater altitudes. The C-47 took the craft to an altitude of 12,000 where free flights back to Rogers Dry Lake began. Pilot for the first series of flights of the M2-F1 was NASA research pilot Milt Thompson. Typical glide flights with the M2-F1 lasted about two minutes and reached speeds of 110 to l20 mph. More than 400 ground tows and 77 aircraft tow flights were carried out with the M2-F1. The success of Dryden's M2-F1 program led to NASA's development and construction of two heavyweight lifting bodies based on studies at NASA's Ames and

  15. Holographic cosmology from a system of M2–M5 branes

    Energy Technology Data Exchange (ETDEWEB)

    Sepehri, Alireza, E-mail: alireza.sepehri@uk.ac.ir [Faculty of Physics, Shahid Bahonar University, P.O. Box 76175, Kerman (Iran, Islamic Republic of); Research Institute for Astronomy and Astrophysics of Maragha (RIAAM), Maragha (Iran, Islamic Republic of); Faizal, Mir, E-mail: f2mir@uwaterloo.ca [Department of Physics and Astronomy, University of Waterloo, Waterloo, ON, N2L 3G1 (Canada); Setare, Mohammad Reza, E-mail: rezakord@ipm.ir [Department of Science, Campus of Bijar, University of Kurdistan, Bijar (Iran, Islamic Republic of); Ali, Ahmed Farag, E-mail: afali@fsu.edu [Department of Physics, Florida State University, Tallahassee, FL 32306 (United States); Department of Physics, Faculty of Science, Benha University, Benha 13518 (Egypt)

    2016-05-15

    In this paper, we analyze the holographic cosmology using a M2–M5 brane configuration. In this configuration, a M2-brane will be placed in between a M5-brane and an anti-M5-brane. The M2-brane will act as a channel for energy to flow from an anti-M5-brane to a M5-brane, and this will increase the degrees of freedom on the M5-brane causing inflation. The inflation will end when the M5-brane and anti-M5-brane get separated. However, at a later stage the distance between the M5-brane and the anti-M5-bran can reduce and this will cause the formation of tachyonic states. These tachyonic states will again open a bridge between the M5-branes and the anti-M5-branes, which will cause further acceleration of the universe.

  16. Holographic cosmology from a system of M2–M5 branes

    International Nuclear Information System (INIS)

    Sepehri, Alireza; Faizal, Mir; Setare, Mohammad Reza; Ali, Ahmed Farag

    2016-01-01

    In this paper, we analyze the holographic cosmology using a M2–M5 brane configuration. In this configuration, a M2-brane will be placed in between a M5-brane and an anti-M5-brane. The M2-brane will act as a channel for energy to flow from an anti-M5-brane to a M5-brane, and this will increase the degrees of freedom on the M5-brane causing inflation. The inflation will end when the M5-brane and anti-M5-brane get separated. However, at a later stage the distance between the M5-brane and the anti-M5-bran can reduce and this will cause the formation of tachyonic states. These tachyonic states will again open a bridge between the M5-branes and the anti-M5-branes, which will cause further acceleration of the universe.

  17. Comments on the Bagger-Lambert theory and multiple M2-branes

    International Nuclear Information System (INIS)

    Raamsdonk, Mark Van

    2008-01-01

    We study the SO(8) superconformal theory proposed recently by Bagger and Lambert as a possible worldvolume theory for multiple M2-branes. For their explicit example with gauge group SO(4), we rewrite the theory (originally formulated in terms of a three-algebra) as an ordinary SU(2) x SU(2) gauge theory with bifundamental matter. In this description, the parity invariance of the theory, required for a proper description of M2-branes, is clarified. We describe the subspace of scalar field configurations on which the potential vanishes, correcting an earlier claim. Finally, we point out, for general three-algebras, a difficulty in constructing the required set of superconformal primary operators which should be present in the correct theory describing multiple M2-branes.

  18. M2 FILTER FOR SPECKLE NOISE SUPPRESSION IN BREAST ULTRASOUND IMAGES

    Directory of Open Access Journals (Sweden)

    E.S. Samundeeswari

    2016-11-01

    Full Text Available Breast cancer, commonly found in women is a serious life threatening disease due to its invasive nature. Ultrasound (US imaging method plays an effective role in screening early detection and diagnosis of Breast cancer. Speckle noise generally affects medical ultrasound images and also causes a number of difficulties in identifying the Region of Interest. Suppressing speckle noise is a challenging task as it destroys fine edge details. No specific filter is designed yet to get a noise free BUS image that is contaminated by speckle noise. In this paper M2 filter, a novel hybrid of linear and nonlinear filter is proposed and compared to other spatial filters with 3×3 kernel size. The performance of the proposed M2 filter is measured by statistical quantity parameters like MSE, PSNR and SSI. The experimental analysis clearly shows that the proposed M2 filter outperforms better than other spatial filters by 2% high PSNR values with regards to speckle suppression.

  19. Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

    Science.gov (United States)

    Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Pelegrín, Pablo

    2016-02-01

    Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis. © Society for Leukocyte Biology.

  20. M2IRAGE: Management of measurements during radiological interventions geographically assisted in the environment

    International Nuclear Information System (INIS)

    Gerphagnon, O.; Roche, H.; Lelache, H.; Guelin, M.; Fauquant, J.M.; Kacenelen, Y.; Armand, Y.

    2010-01-01

    This report presents the M 2 IRAGE software, a data processing tool designed to share radioactivity measurements and to give a schematised view of a radiological situation and of its evolution, while respecting different legal frameworks, notably the obligation to produce a radiological measurement programme. After a simplified recall of the crisis management organisation, the authors describe the M 2 IRAGE software and hardware architecture, the functions of its main modules (presentation of radioprotection information during field intervention, field mission management, data browsing, and data transmission to field teams). While giving some display examples, the authors describe how an event is managed and processed by this tool: event creation, measurement acquisition, aid to decision, team management. They report and discuss the results of a national exercise which took place in September 2009 in Saclay with a prototype version of M 2 IRAGE

  1. Isoliquiritigenin, a flavonoid from licorice, blocks M2 macrophage polarization in colitis-associated tumorigenesis through downregulating PGE{sub 2} and IL-6

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Haixia [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zhang, Xinhua [Department of Liver, Biliary And Pancreatic Tumors, Hubei Cancer Hospital, Wuhan 430079 (China); Chen, Xuewei; Li, Ying; Ke, Zunqiong [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Tang, Tian [Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Guo, Austin M. [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Department of Pharmacology, New York Medical College, Valhalla, NY 10595 (United States); Chen, Honglei, E-mail: hl-chen@whu.edu.cn [Department of Pathology and Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China)

    2014-09-15

    M2 macrophage polarization is implicated in colorectal cancer development. Isoliquiritigenin (ISL), a flavonoid from licorice, has been reported to prevent azoxymethane (AOM) induced colon carcinogenesis in animal models. Here, in a mouse model of colitis-associated tumorigenesis induced by AOM/dextran sodium sulfate (DSS), we investigated the chemopreventive effects of ISL and its mechanisms of action. Mice were treated with AOM/DSS and randomized to receive either vehicle or ISL (3, 15 and 75 mg/kg). Tumor load, histology, immunohistochemistry, and gene and protein expressions were determined. Intragastric administration of ISL for 12 weeks significantly decreased colon cancer incidence, multiplicity and tumor size by 60%, 55.4% and 42.6%, respectively. Moreover, ISL inhibited M2 macrophage polarization. Such changes were accompanied by downregulation of PGE{sub 2} and IL-6 signaling. Importantly, depletion of macrophages by clodronate (Clod) or zoledronic acid (ZA) reversed the effects of ISL. In parallel, in vitro studies also demonstrated that ISL limited the M2 polarization of RAW264.7 cells and mouse peritoneal macrophages with concomitant inactivation of PGE{sub 2}/PPARδ and IL-6/STAT3 signaling. Conversely, exogenous addition of PGE{sub 2} or IL-6, or overexpression of constitutively active STAT3 reversed ISL-mediated inhibition of M2 macrophage polarization. In summary, dietary flavonoid ISL effectively inhibits colitis-associated tumorigenesis through hampering M2 macrophage polarization mediated by the interplay between PGE{sub 2} and IL-6. Thus, inhibition of M2 macrophage polarization is likely to represent a promising strategy for chemoprevention of colorectal cancer. - Highlights: • Isoliquiritigenin (ISL) prevents colitis-associated tumorigenesis. • ISL inhibits M2 macrophage polarization in vivo and in vitro. • ISL inhibits PGE{sub 2} and IL-6 signaling in colitis-associated tumorigenesis. • ISL may be an attractive candidate agent for

  2. Control software architecture and operating modes of the Model M-2 maintenance system

    Energy Technology Data Exchange (ETDEWEB)

    Satterlee, P.E. Jr.; Martin, H.L.; Herndon, J.N.

    1984-04-01

    The Model M-2 maintenance system is the first completely digitally controlled servomanipulator. The M-2 system allows dexterous operations to be performed remotely using bilateral force-reflecting master/slave techniques, and its integrated operator interface takes advantage of touch-screen-driven menus to allow selection of all possible operating modes. The control system hardware for this system has been described previously. This paper describes the architecture of the overall control system. The system's various modes of operation are identified, the software implementation of each is described, system diagnostic routines are described, and highlights of the computer-augmented operator interface are discussed. 3 references, 5 figures.

  3. Modelling the enigmatic Late Pliocene Glacial Event - Marine Isotope Stage M2

    Science.gov (United States)

    Dolan, Aisling M.; Haywood, Alan M.; Hunter, Stephen J.; Tindall, Julia C.; Dowsett, Harry J.; Hill, Daniel J.; Pickering, Steven J.

    2015-01-01

    The Pliocene Epoch (5.2 to 2.58 Ma) has often been targeted to investigate the nature of warm climates. However, climate records for the Pliocene exhibit significant variability and show intervals that apparently experienced a cooler than modern climate. Marine Isotope Stage (MIS) M2 (~ 3.3 Ma) is a globally recognisable cooling event that disturbs an otherwise relatively (compared to present-day) warm background climate state. It remains unclear whether this event corresponds to significant ice sheet build-up in the Northern and Southern Hemisphere. Estimates of sea level for this interval vary, and range from modern values to estimates of 65 m sea level fall with respect to present day. Here we implement plausible M2 ice sheet configurations into a coupled atmosphere–ocean climate model to test the hypothesis that larger-than-modern ice sheet configurations may have existed at M2. Climate model results are compared with proxy climate data available for M2 to assess the plausibility of each ice sheet configuration. Whilst the outcomes of our data/model comparisons are not in all cases straight forward to interpret, there is little indication that results from model simulations in which significant ice masses have been prescribed in the Northern Hemisphere are incompatible with proxy data from the North Atlantic, Northeast Arctic Russia, North Africa and the Southern Ocean. Therefore, our model results do not preclude the possibility of the existence of larger ice masses during M2 in the Northern or Southern Hemisphere. Specifically they are not able to discount the possibility of significant ice masses in the Northern Hemisphere during the M2 event, consistent with a global sea-level fall of between 40 m and 60 m. This study highlights the general need for more focused and coordinated data generation in the future to improve the coverage and consistency in proxy records for M2, which will allow these and future M2 sensitivity tests to be interrogated

  4. Complete Suppression of the m=2/n=1 NTM Using ECCD on DIII-D

    International Nuclear Information System (INIS)

    Petty, C.C.; La Haye, R.J.; Luce, T.C.; Humphreys, D.A.; Lohr, J.; Prater, R.; Austin, M.E.; Harvey, R.W.; Wade, M.R.

    2003-01-01

    Complete suppression of the m=2/n=1 neoclassical tearing mode (NTM) is reported for the first time using electron cyclotron current drive (ECCD) to noninductively generate current at the radius of the island O-point. Experiments on the DIII-D tokamak show that the maximum shrinkage of the m=2/n=1 island amplitude occurs when the ECCD location coincides with the q=2 surface. Estimates of the ECCD radial profile width from the island shrinkage are consistent with ray tracing calculations but may allow for a factor-of-1.5 broadening from electron radial transport

  5. Control software architecture and operating modes of the Model M-2 maintenance system

    International Nuclear Information System (INIS)

    Satterlee, P.E. Jr.; Martin, H.L.; Herndon, J.N.

    1984-04-01

    The Model M-2 maintenance system is the first completely digitally controlled servomanipulator. The M-2 system allows dexterous operations to be performed remotely using bilateral force-reflecting master/slave techniques, and its integrated operator interface takes advantage of touch-screen-driven menus to allow selection of all possible operating modes. The control system hardware for this system has been described previously. This paper describes the architecture of the overall control system. The system's various modes of operation are identified, the software implementation of each is described, system diagnostic routines are described, and highlights of the computer-augmented operator interface are discussed. 3 references, 5 figures

  6. [Analytic study of dot blotting for the detection of anti-Jo-1, anti-M2, anti-ribosomes and anti-LKM].

    Science.gov (United States)

    Huguet, S; Sghiri, R; Ballot, E; Johanet, C

    2004-01-01

    The Cyto-Dot 4 HM043 kit commercialised by BMD, has replaced the Cyto-Dot HM010 kit that allowed three auto-antibodies detection (anti-Jo-1, anti-M2 and anti-ribosomal protein). Detection of anti-LKM1 auto-antibody was added. These four auto-antibodies have in common only the intracytoplasmic localisation of their respective antigen. The aim of our study was to evaluate this new kit using 104 sera and to compare our results with reference techniques (indirect immunofluorescence IF for anti-M2, anti-ribosomal protein and anti-LKM1, double immunodiffusion ID for anti-Jo-1 and anti-LKM1, western blotting WB for anti-M2) and with Cyto-Dot HM010. The one hundred and four sera were divided into five groups: Group I (n = 12) with anti-Jo-1 detected by ID; Group II (n = 28) with 26 anti-M2 positive by IF and WB, 2 anti-M2 positive only by WB; Group III (n = 10) with anti-ribosomal protein detected by IF 5 of which precipitated by ID; Group IV (n = 32) with anti-LKM1 by IF and ID divided into 18 AIH2 and 14 HCV; Group V (n = 22) consisting of 14 healthy individuals and 8 patients with hypergammaglobulinemia. Results of this study are similar to those of Cyto-Dot HM010 for the three auto-antibodies already in use. Cyto-Dot 4 is a very good anti-LKM1 confirmation method as it is ID. Copyright John Libbey Eurotext 2003.

  7. Effects of ingesting protein with various forms of carbohydrate following resistance-exercise on substrate availability and markers of anabolism, catabolism, and immunity

    Directory of Open Access Journals (Sweden)

    Greenwood Michael

    2007-11-01

    Full Text Available Abstract Background Ingestion of carbohydrate (CHO and protein (PRO following intense exercise has been reported to increase insulin levels, optimize glycogen resynthesis, enhance PRO synthesis, and lessen the immuno-suppressive effects of intense exercise. Since different forms of CHO have varying glycemic effects, the purpose of this study was to determine whether the type of CHO ingested with PRO following resistance-exercise affects blood glucose availability and insulin levels, markers of anabolism and catabolism, and/or general immune markers. Methods 40 resistance-trained subjects performed a standardized resistance training workout and then ingested in a double blind and randomized manner 40 g of whey PRO with 120 g of sucrose (S, honey powder (H, or maltodextrin (M. A non-supplemented control group (C was also evaluated. Blood samples were collected prior to and following exercise as well as 30, 60, 90, and 120 min after ingestion of the supplements. Data were analyzed by repeated measures ANOVA or ANCOVA using baseline values as a covariate if necessary. Results Glucose concentration 30 min following ingestion showed the H group (7.12 ± 0.2 mmol/L to be greater than S (5.53 ± 0.6 mmol/L; p uIU/mL, H (150.1 ± 25.39 uIU/mL, and M (154.8 ± 18.9 uIU/mL were greater than C (8.7 ± 2.9 uIU/mL as was AUC with no significant differences observed among types of CHO. No significant group × time effects were observed among groups in testosterone, cortisol, the ratio of testosterone to cortisol, muscle and liver enzymes, or general markers of immunity. Conclusion CHO and PRO ingestion following exercise significantly influences glucose and insulin concentrations. Although some trends were observed suggesting that H maintained blood glucose levels to a better degree, no significant differences were observed among types of CHO ingested on insulin levels. These findings suggest that each of these forms of CHO can serve as effective sources of

  8. From WSN towards WoT: Open API Scheme Based on oneM2M Platforms

    Science.gov (United States)

    Kim, Jaeho; Choi, Sung-Chan; Ahn, Il-Yeup; Sung, Nak-Myoung; Yun, Jaeseok

    2016-01-01

    Conventional computing systems have been able to be integrated into daily objects and connected to each other due to advances in computing and network technologies, such as wireless sensor networks (WSNs), forming a global network infrastructure, called the Internet of Things (IoT). To support the interconnection and interoperability between heterogeneous IoT systems, the availability of standardized, open application programming interfaces (APIs) is one of the key features of common software platforms for IoT devices, gateways, and servers. In this paper, we present a standardized way of extending previously-existing WSNs towards IoT systems, building the world of the Web of Things (WoT). Based on the oneM2M software platforms developed in the previous project, we introduce a well-designed open API scheme and device-specific thing adaptation software (TAS) enabling WSN elements, such as a wireless sensor node, to be accessed in a standardized way on a global scale. Three pilot services are implemented (i.e., a WiFi-enabled smart flowerpot, voice-based control for ZigBee-connected home appliances, and WiFi-connected AR.Drone control) to demonstrate the practical usability of the open API scheme and TAS modules. Full details on the method of integrating WSN elements into three example systems are described at the programming code level, which is expected to help future researchers in integrating their WSN systems in IoT platforms, such as oneM2M. We hope that the flexibly-deployable, easily-reusable common open API scheme and TAS-based integration method working with the oneM2M platforms will help the conventional WSNs in diverse industries evolve into the emerging WoT solutions. PMID:27782058

  9. From WSN towards WoT: Open API Scheme Based on oneM2M Platforms

    Directory of Open Access Journals (Sweden)

    Jaeho Kim

    2016-10-01

    Full Text Available Conventional computing systems have been able to be integrated into daily objects and connected to each other due to advances in computing and network technologies, such as wireless sensor networks (WSNs, forming a global network infrastructure, called the Internet of Things (IoT. To support the interconnection and interoperability between heterogeneous IoT systems, the availability of standardized, open application programming interfaces (APIs is one of the key features of common software platforms for IoT devices, gateways, and servers. In this paper, we present a standardized way of extending previously-existing WSNs towards IoT systems, building the world of the Web of Things (WoT. Based on the oneM2M software platforms developed in the previous project, we introduce a well-designed open API scheme and device-specific thing adaptation software (TAS enabling WSN elements, such as a wireless sensor node, to be accessed in a standardized way on a global scale. Three pilot services are implemented (i.e., a WiFi-enabled smart flowerpot, voice-based control for ZigBee-connected home appliances, and WiFi-connected AR.Drone control to demonstrate the practical usability of the open API scheme and TAS modules. Full details on the method of integrating WSN elements into three example systems are described at the programming code level, which is expected to help future researchers in integrating their WSN systems in IoT platforms, such as oneM2M. We hope that the flexibly-deployable, easily-reusable common open API scheme and TAS-based integration method working with the oneM2M platforms will help the conventional WSNs in diverse industries evolve into the emerging WoT solutions.

  10. From WSN towards WoT: Open API Scheme Based on oneM2M Platforms.

    Science.gov (United States)

    Kim, Jaeho; Choi, Sung-Chan; Ahn, Il-Yeup; Sung, Nak-Myoung; Yun, Jaeseok

    2016-10-06

    Conventional computing systems have been able to be integrated into daily objects and connected to each other due to advances in computing and network technologies, such as wireless sensor networks (WSNs), forming a global network infrastructure, called the Internet of Things (IoT). To support the interconnection and interoperability between heterogeneous IoT systems, the availability of standardized, open application programming interfaces (APIs) is one of the key features of common software platforms for IoT devices, gateways, and servers. In this paper, we present a standardized way of extending previously-existing WSNs towards IoT systems, building the world of the Web of Things (WoT). Based on the oneM2M software platforms developed in the previous project, we introduce a well-designed open API scheme and device-specific thing adaptation software (TAS) enabling WSN elements, such as a wireless sensor node, to be accessed in a standardized way on a global scale. Three pilot services are implemented (i.e., a WiFi-enabled smart flowerpot, voice-based control for ZigBee-connected home appliances, and WiFi-connected AR.Drone control) to demonstrate the practical usability of the open API scheme and TAS modules. Full details on the method of integrating WSN elements into three example systems are described at the programming code level, which is expected to help future researchers in integrating their WSN systems in IoT platforms, such as oneM2M. We hope that the flexibly-deployable, easily-reusable common open API scheme and TAS-based integration method working with the oneM2M platforms will help the conventional WSNs in diverse industries evolve into the emerging WoT solutions.

  11. An analytical study of M2 tidal waves in the Taiwan Strait using an extended Taylor method

    Science.gov (United States)

    Wu, Di; Fang, Guohong; Cui, Xinmei; Teng, Fei

    2018-02-01

    The tides in the Taiwan Strait (TS) feature large semidiurnal lunar (M2) amplitudes. An extended Taylor method is employed in this study to provide an analytical model for the M2 tide in the TS. The strait is idealized as a rectangular basin with a uniform depth, and the Coriolis force and bottom friction are retained in the governing equations. The observed tides at the northern and southern openings are used as open boundary conditions. The obtained analytical solution, which consists of a stronger southward propagating Kelvin wave, a weaker northward propagating Kelvin wave, and two families of Poincaré modes trapped at the northern and southern openings, agrees well with the observations in the strait. The superposition of two Kelvin waves basically represents the observed tidal pattern, including an anti-nodal band in the central strait, and the cross-strait asymmetry (greater amplitudes in the west and smaller in the east) of the anti-nodal band. Inclusion of Poincaré modes further improves the model result in that the cross-strait asymmetry can be better reproduced. To explore the formation mechanism of the northward propagating wave in the TS, three experiments are carried out, including the deep basin south of the strait. The results show that the southward incident wave is reflected to form a northward wave by the abruptly deepened topography south of the strait, but the reflected wave is slightly weaker than the northward wave obtained from the above analytical solution, in which the southern open boundary condition is specified with observations. Inclusion of the forcing at the Luzon Strait strengthens the northward Kelvin wave in the TS, and the forcing is thus of some (but lesser) importance to the M2 tide in the TS.

  12. Models of the Mass-ejection Histories of Pre-planetary Nebulae. II. The Formation of Minkowski’s Butterfly and its Proboscis in M2–9

    Science.gov (United States)

    Balick, Bruce; Frank, Adam; Liu, Baowei; Corradi, Romano

    2018-02-01

    M2–9, or the “Minkowski’s Butterfly,” is one of the most iconic outflow sources from an evolved star. In this paper we present a hydrodynamic model of M2–9 in which the nebula is formed and shaped by a steady, low-density (“light”), mildly collimated “spray” of gas injected at 200 km s‑1 that interacts with a far denser, intrinsically simple pre-existing AGB wind that has slowly formed all of the complex features within M2–9’s lobes (including the knot pairs N3/S3 and N4/S4 at their respective leading edges, and the radial gradient of Doppler shifts within 20″ of the nucleus). We emphasize that the knot pairs are not ejected from the star but formed in situ. In addition, the observed radial speed of the knots is only indirectly related to the speed of the gas injected by the star. The model allows us to probe the early history of the wind geometry and lobe formation. We also formulate a new estimate of the nebular distance D = 1.3 kpc. The physical mechanism that accounts for the linear radial speed gradient in M2–9 applies generally to many other pre-planetary nebulae whose hollow lobes exhibit similar gradients along their edges.

  13. M2-F1 in flight over lakebed on tow line

    Science.gov (United States)

    1963-01-01

    After initial ground-tow flights of the M2-F1 using the Pontiac as a tow vehicle, the way was clear to make air tows behind a C-47. The first air tow took place on 16 August 1963. Pilot Milt Thompson found that the M2-F1 flew well, with good control. This first flight lasted less than two minutes from tow-line release to touchdown. The descent rate was 4,000 feet per minute. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got

  14. Investigation into adamantane-based M2 inhibitors with FB-QSAR.

    Science.gov (United States)

    Wei, Hang; Wang, Cheng-Hua; Du, Qi-Shi; Meng, Jianzong; Chou, Kuo-Chen

    2009-07-01

    Because of their high resistance rate to the existing drugs, influenza A viruses have become a threat to human beings. It is known that the replication of influenza A viruses needs a pH-gated proton channel, the so-called M2 channel. Therefore, to develop effective drugs against influenza A, the most logic strategy is to inhibit the M2 channel. Recently, the atomic structure of the M2 channel was determined by NMR spectroscopy (Schnell, J.R. and Chou, J.J., Nature, 2008, 451, 591-595). The high-resolution NMR structure has provided a solid basis for structure-based drug design approaches. In this study, a benchmark dataset has been constructed that contains 34 newly-developed adamantane-based M2 inhibitors and covers considerable structural diversities and wide range of bioactivities. Based on these compounds, an in-depth analysis was performed with the newly developed fragment-based quantitative structure-activity relationship (FB-QSAR) algorithm. The results thus obtained provide useful insights for dealing with the drug-resistant problem and designing effective adamantane-based antiflu drugs.

  15. Massive M2M Access with Reliability Guarantees in LTE Systems

    DEFF Research Database (Denmark)

    Madueño, Germán Corrales; Kiilerich Pratas, Nuno; Stefanovic, Cedomir

    2015-01-01

    Machine-to-Machine (M2M) communications are one of the major drivers of the cellular network evolution towards 5G systems. One of the key challenges is on how to provide reliability guarantees to each accessing device in a situation in which there is a massive number of almost-simultaneous arriva...

  16. Initial search for triggered gamma emission from Hf-178(m2) using the YSU miniball array

    Czech Academy of Sciences Publication Activity Database

    Carroll, J. J.; Burnett, J.; Drummond, T.; Lepak, J.; Propri, R.; Smith, D.; Karamian, S. A.; Adam, Jindřich; Stedile, F.; Agee, FJ.

    2002-01-01

    Roč. 143, 1, 2, 3, 4 (2002), s. 37-54 ISSN 0304-3843 Institutional research plan: CEZ:AV0Z1048901 Keywords : triggered gamma emission * Hf-178(m2) * nuclear batteries Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 0.533, year: 2002

  17. Almost optimal distributed M2M multicasting in wireless mesh networks

    DEFF Research Database (Denmark)

    Xin, Qin; Manne, Fredrik; Zhang, Yan

    2012-01-01

    Wireless Mesh Networking (WMN) is an emerging communication paradigm to enable resilient, cost-efficient and reliable services for the future-generation wireless networks. In this paper, we study the problem of multipoint-to- multipoint (M2M) multicasting in a WMN which aims to use the minimum nu...

  18. Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages

    Science.gov (United States)

    Waqas, Syed F. Hassnain; Hoang, Anh Cuong; Ampem, Grace; Azegrouz, Hind; Balogh, Lajos; Thuróczy, Julianna; Gerling, Ivan C.; Nam, Sorim; Lim, Jong-Seok; Martinez-Ibañez, Juncal; Real, José T.; Paschke, Stephan; Quillet, Raphaëlle; Ayachi, Safia; Simonin, Frédéric; Schneider, E. Marion; Brinkman, Jacqueline A.; Seroogy, Christine M.

    2017-01-01

    The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet–fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage–associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs. PMID:28581443

  19. Formation of the high-spin Hf-179m2 isomer in reactor irradiations

    Czech Academy of Sciences Publication Activity Database

    Karamian, S. A.; Carroll, J. J.; Adam, Jindřich; Kulagin, EN.; Shabalin, EP.

    2004-01-01

    Roč. 14, č. 4 (2004), s. 438-441 ISSN 1054-660X R&D Projects: GA MŠk(CZ) ME 134 Keywords : reactor irradiation * high-spin Hf-179m2 Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 0.836, year: 2004

  20. Comparison of Plasma Tu-M2-PK and CA19-9 in Pancreatic Cancer

    DEFF Research Database (Denmark)

    Joergensen, Maiken Thyregod; Heegaard, Niels H H; Schaffalitzky de Muckadell, Ove B

    2009-01-01

    because of suspicion of pancreatic cancer. Of these, 51 patients had their conditions diagnosed as PDAC, whereas this diagnosis was ruled out in 52 after 12 months of follow-up. The performance of Tu-M2-PK was compared with that of CA19-9 using cutoff values 15 and 37 U/mL, respectively. RESULTS...

  1. M2-F1 mounted in NASA Ames Research Center 40x80 foot wind tunnel

    Science.gov (United States)

    1962-01-01

    After the first attempted ground-tow tests of the M2-F1 in March 1963, the vehicle was taken to the Ames Research Center, Mountain View, CA, for wind-tunnel testing. During these tests, Milt Thompson and others were in the M2-F1 to position the control surfaces for each test. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got the M2-F1 airborne to prove it could fly safely and to train pilots before they were towed behind a C

  2. A Survey on M2M Systems for mHealth: A Wireless Communications Perspective

    Directory of Open Access Journals (Sweden)

    Elli Kartsakli

    2014-09-01

    Full Text Available In the new era of connectivity, marked by the explosive number of wireless electronic devices and the need for smart and pervasive applications, Machine-to-Machine (M2M communications are an emerging technology that enables the seamless device interconnection without the need of human interaction. The use of M2M technology can bring to life a wide range of mHealth applications, with considerable benefits for both patients and healthcare providers. Many technological challenges have to be met, however, to ensure the widespread adoption of mHealth solutions in the future. In this context, we aim to provide a comprehensive survey on M2M systems for mHealth applications from a wireless communication perspective. An end-to-end holistic approach is adopted, focusing on different communication aspects of the M2M architecture. Hence, we first provide a systematic review ofWireless Body Area Networks (WBANs, which constitute the enabling technology at the patient’s side, and then discuss end-to-end solutions that involve the design and implementation of practical mHealth applications. We close the survey by identifying challenges and open research issues, thus paving the way for future research opportunities.

  3. Growth and properties of M2-xCexCuO4+d single crystals

    International Nuclear Information System (INIS)

    Matacotta, F.C.; Morales de La Garza, L.; Nevriva, M.; Nardin, G.; Randaccio, L.; Zangrando, E.

    1989-10-01

    The M 2-x Ce x CuO 4+d crystals where M is a rare earth ion (like Nd, Pr etc.) were prepared by a modified flux method, their structure has been investigated by scanning electron microscopy and energy dispersive X-ray analysis and the electrical resistivity was measured on a number of crystals. 5 refs, 3 figs, 3 tabs

  4. Efficient LTE Access with Collision Resolution for Massive M2M Communications

    DEFF Research Database (Denmark)

    Madueño, Germán Corrales; Stefanovic, Cedomir; Popovski, Petar

    2014-01-01

    outage. In this work we propose a LTE RACH scheme tailored for delay-sensitive M2M services with synchronous traffic arrivals. The key idea is, upon detection of a RACH overload, to apply a collision resolution algorithm based on splitting trees. The solution is implemented on top of the existing LTE...

  5. Macrophages during the fibrotic process: M2 as friend and foe.

    Directory of Open Access Journals (Sweden)

    Tarcio Teodoro Braga

    2015-11-01

    Full Text Available Macrophages play essential activities in homeostasis maintenance, tissue regeneration and wound healing. However, when the physiological process of wound healing is deregulated by persistent insults and chronic diseases, macrophages can participate actively in the development of fibrosis. In this regard, the exacerbation or resolution of fibrosis depends on the type of macrophages polarized and the severity and duration of the inflammatory insult. M1 macrophages use glycolytic metabolism to optimize oxygen consumption and activate myofibroblasts and fibrocytes. On the other hand, M2 macrophages, which use oxidative metabolism, have anti-inflammatory properties due to their capacity to produce and secrete IL-10, TGFβ and arginase that promotes tissue repair. However, when the primary insult is not controlled and there is a persistent M2 macrophage activity, these cells promote ECM deposition through the continuous production of TGFβ and growth factors. In this scenario, M2 macrophages act as a break point between normal wound healing and the pro-fibrotic process. Here, we review the aspects of tissue repair based on macrophage biology and we evidence scar formation is directly related to the degree of inflammation, but also with the appearance of M2 macrophages.

  6. Multiple promoters drive tissue-specific expression of the human M2 muscarinic acetylcholine receptor gene

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Alena; Bruce, A. W.; Doležal, Vladimír; Tuček, Stanislav; Buckley, N. J.

    2004-01-01

    Roč. 91, č. 1 (2004), s. 88-98 ISSN 0022-3042 R&D Projects: GA AV ČR IAA5011306 Institutional research plan: CEZ:AV0Z5011922 Keywords : M2 muscarinic receptor * neuron-restrictive silence factor * promoter Subject RIV: ED - Physiology Impact factor: 4.824, year: 2004

  7. Thermoelectric Performance of the MXenes M2CO2 (M = Ti, Zr, or Hf)

    KAUST Repository

    Gandi, Appala; Alshareef, Husam N.; Schwingenschlö gl, Udo

    2016-01-01

    MXenes, M2CO2, where M = Ti, Zr, or Hf, in order to evaluate the effect of the metal M on the thermoelectric performance. The lattice contribution to the thermal conductivity, obtained from the phonon life times, is found to be lowest in Ti2CO2

  8. Exploiting the Capture Effect to Enhance RACH Performance in Cellular-Based M2M Communications

    Directory of Open Access Journals (Sweden)

    Jonghun Kim

    2017-09-01

    Full Text Available Cellular-based machine-to-machine (M2M communication is expected to facilitate services for the Internet of Things (IoT. However, because cellular networks are designed for human users, they have some limitations. Random access channel (RACH congestion caused by massive access from M2M devices is one of the biggest factors hindering cellular-based M2M services because the RACH congestion causes random access (RA throughput degradation and connection failures to the devices. In this paper, we show the possibility exploiting the capture effects, which have been known to have a positive impact on the wireless network system, on RA procedure for improving the RA performance of M2M devices. For this purpose, we analyze an RA procedure using a capture model. Through this analysis, we examine the effects of capture on RA performance and propose an Msg3 power-ramping (Msg3 PR scheme to increase the capture probability (thereby increasing the RA success probability even when severe RACH congestion problem occurs. The proposed analysis models are validated using simulations. The results show that the proposed scheme, with proper parameters, further improves the RA throughput and reduces the connection failure probability, by slightly increasing the energy consumption. Finally, we demonstrate the effects of coexistence with other RA-related schemes through simulation results.

  9. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

    Directory of Open Access Journals (Sweden)

    María Carolina Ortiz

    Full Text Available Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ or alternative-M2 (M2-MФ activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1, widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.

  10. Construction of Δm2--sin2 2θ plots

    International Nuclear Information System (INIS)

    Snyder, R.

    1991-01-01

    In the two-flavor approximation, the probability for a neutrino to oscillate from one flavor to the other is given by Ρ νa → νb = sin 2 2θ sin 2 (1.27 Δm 2 L/E ν ) where θ is the mixing angle, Δm 2 = |m νa 2 - m νb 2 | is measured in (eV/c 2 ) 2 , L, the distance from the source, is measured in km, and E, the beam energy, is measured in GeV. If either Δm 2 or sin 2 2Θ is zero, there is no oscillation. They might also have small, non-zero values, causing the oscillations to be so small as to be unobservable in a particular experiment. They may also have values which allow us to determine the probability of oscillation, but so far no compelling evidence for oscillation exists. The universal method of portraying what region of parameter space is explored by a neutrino oscillation experiment is to mark off an area on a Δm 2 vs. sin 2 2θ plot. Typically, a line is graphed, with the claim that if the experiment finds not evidence of oscillation, one can, for example, be 90% certain that Δm 2 and sin 2 2θ lie within the region below and to the left of the line. Since these plots are so widely used, it is useful to understand the process by which they are created

  11. A boosting skin vaccination with dissolving microneedle patch encapsulating M2e vaccine broadens the protective efficacy of conventional influenza vaccines.

    Science.gov (United States)

    Zhu, Wandi; Pewin, Winston; Wang, Chao; Luo, Yuan; Gonzalez, Gilbert X; Mohan, Teena; Prausnitz, Mark R; Wang, Bao-Zhong

    2017-09-10

    The biodegradable microneedle patch (MNP) is a novel technology for vaccine delivery that could improve the immunogenicity of vaccines. To broaden the protective efficiency of conventional influenza vaccines, a new 4M2e-tFliC fusion protein construct containing M2e sequences from different subtypes was generated. Purified fusion protein was encapsulate into MNPs with a biocompatible polymer for use as a boosting vaccine. The results demonstrated that mice receiving a conventional inactivated vaccine followed by a skin-applied dissolving 4M2e-tFliC MNP boost could better maintain the humoral antibody response than that by the conventional vaccine-prime alone. Compared with an intramuscular injection boost, mice receiving the MNP boost showed significantly enhanced cellular immune responses, hemagglutination-inhibition (HAI) titers, and neutralization titers. Increased frequency of antigen-specific plasma cells and long-lived bone marrow plasma cells was detected in the MNP boosted group as well, indicating that skin vaccination with 4M2e-tFliC facilitated a long-term antibody-mediated immunity. The 4M2e-tFliC MNP-boosted group also possessed enhanced protection against high lethal dose challenges against homologous A/PR/8/34 and A/Aichi/2/68 viruses and protection for a majority of immunized mice against a heterologous A/California/07/2009 H1N1 virus. High levels of M2e specific immune responses were observed in the 4M2e-tFliC MNP-boosted group as well. These results demonstrate that a skin-applied 4M2e-tFliC MNP boosting immunization to seasonal vaccine recipients may be a rapid approach for increasing the protective efficacy of seasonal vaccines in response to a significant drift seen in circulating viruses. The results also provide a new perspective for future exploration of universal influenza vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Structure of vanadium oxosulfato complexes in V2O5-M2S2O7-M2SO4 (M = K, Cs) melts. A high temperature spectroscopic study

    DEFF Research Database (Denmark)

    Boghosian, S.; Chrissanthopoulos, A.; Fehrmann, Rasmus

    2002-01-01

    2 atmosphere (P-SO2 = 0-1.2 atm). The data are in agreement with the V-V V-IV equilibrium: (VO)(2)O(SO4)(4)(4-)(1) + SO2(g) 2VO(SO4)(2)(2-)(1) + SO3(g). SO2 does not coordinate to the V-V complex but starts significantly to coordinate to V-IV for P-SO2 > 0.4 atm according to VO(SO4)(2)(2-)(1) + SO2......(g) VO(SO4)(2)SO22-(1). The Raman spectral features and the exploitation of the relative Raman intensities indicate that the (VO)(2)O(SO4)(4)(4-) dimeric complex unit, possessing a V-O-V bridge, is formed in the V2O5-M2S2O7 binary mixtures. The spectral changes occurring upon interaction...

  13. M2-F1 in flight during low-speed car tow

    Science.gov (United States)

    1963-01-01

    The M2-F1 shown in flight during a low-speed car tow runs across the lakebed. Such tests allowed about two minutes to test the vehicle's handling in flight. NASA Flight Research Center (later redesignated the Dryden Flight Research Center) personnel conducted as many as 8 t