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Sample records for m2 protein forms

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

    Directory of Open Access Journals (Sweden)

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

    DEFF Research Database (Denmark)

    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......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...... 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...

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

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

    Science.gov (United States)

    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.

  5. Solid State NMR Observation of Phenylalanine Residues in M2 Protein from Influenza a Virus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ The M2 protein from influenza A functions as a proton channel. It has been cloned and over-expressed in Escherchia coli. Large quantities of recombinant protein are purified by Ni2 affinity chromatography. The residues in M2 have been selectively labeled with 15N in an aromatic amino acid autotroph CT19.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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......e 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...

  7. Fusion to chicken C3d enhances the immunogenicity of the M2 protein of avian influenza virus

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    Zhang Li

    2010-05-01

    Full Text Available Abstract Background Current vaccines to avian influenzae virus (AIV, a highly contagious disease of birds, need to be constantly updated due to the high level of variation in the target antigens. Therefore, a vaccine that could induce broad cross protection against AIV is required. The M2 membrane protein is structurally conserved amongst AIV subtypes but tends in induce a poor immune response, whereas C3d has been shown in many species to enhance immunogenicity. In this study, we investigated the potential of M2-avian C3d fusion proteins to provide effective immunity. Results We fused chicken complement C3d to sM2 (M2 protein with the transmembrane region deleted of AIV and expressed four fusion proteins, GST (Glutathione S-transferase tagged proteins in pGEX expression vector -C3d-sM2, GST-C3d-L2-sM2, GST-C3d-L1-C3d-sM2 and GST-C3d-L1-C3d-L2-sM2 were used to immunize mice. In addition, Specific pathogen free (SPF chickens were inoculated with the plasmids pcDNA-sM2, pcDNA-C3d-L1-C3d-L2-sM2, GST-sM2 and GST-C3d-L1-C3d-L2-sM2. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA for sM2 antibody, and all the test animals were challenged with A/chicken/Bei Jing/WD9/98 (H9N2 virus. Results revealed that the anti-sM2 antibody in mice and chickens vaccinated with these proteins was higher than the nonfused forms of sM2, the GST-C3d-L1-C3d-L2-sM2 groups have conferred the highest 30% and 20% protection ratio in mice and chickens respectively. In addition, the pcDNA-C3d-L1-C3d-L2-sM2 also enhances the antibody responses to sM2 compared to pcDNA-sM2 in chickens, and acquired 13.3% protection ratio. Conclusion These results indicated that chicken C3d enhanced the humoral immunity against AIV M2 protein either fused proteins expressed by the prokaryotic system or with the DNA vaccine. Nevertheless, in view of the poor protection ratio for these animals, we speculated that this is not a worthy developing of vaccine in these

  8. M2e通用流感疫苗的研究进展%Universal influenza vaccine based on the extracellular domain of M2 protein

    Institute of Scientific and Technical Information of China (English)

    花艳红; 王希良

    2009-01-01

    M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.%Matrix protein M2 (M2) is the membrane protein of Influenza A with an extracellular domain of 24 amino acid residues, which is strongly conserved across virus strains. M2 plays an important role in the life cy-cle of the Influenza A virus and has been the target of antiviral drugs. IgG subtype antibodies directed against M2e can prevent death from influenza and reduce morbidity in animal models for influenza disease. This review summarizes the findings on M2e vaccine candidates and addresses some key questions about this Influenza A vaccine target.

  9. Form factors of descendant operators: Reduction to perturbed $M(2,2s+1)$ models

    CERN Document Server

    Lashkevich, Michael

    2014-01-01

    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 $\\Phi_{13}$\\=/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 $T_{\\pm2k}$, $\\Theta_{\\pm(2k-2)}$, which correspond to the spin $\\pm(2k-1)$ 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 $T\\bar T$ operator. The construction is analytic in the $s$ parameter and, therefore, makes sense in the sine-Gordon theory.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lashkevich, Michael [Landau Institute for Theoretical Physics,1a prospekt Akademika Semenova, 142432 Chernogolovka (Russian Federation); Moscow Institute of Physics and Technology,9 Institutsky per., 141707 Dolgoprudny (Russian Federation); Kharkevich Institute for Information Transmission Problems,19 Bolshoy Karetny per., 127994 Moscow (Russian Federation); Pugai, Yaroslav [Landau Institute for Theoretical Physics,1a prospekt Akademika Semenova, 142432 Chernogolovka (Russian Federation); Moscow Institute of Physics and Technology,9 Institutsky per., 141707 Dolgoprudny (Russian Federation)

    2015-04-23

    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{sub 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{sub ±2k}, Θ{sub ±(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{sub 2k}T{sub −2l}, which generalize the famous TT̄ operator. The construction is analytic in the s parameter and, therefore, makes sense in the sine-Gordon theory.

  11. Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation.

    Science.gov (United States)

    Parks, G D; Hull, J D; Lamb, R A

    1989-11-01

    The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.

  12. 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

    2008-04-01

    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

  13. 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

  14. Study on phylogenetic relationships, variability, and correlated mutations in M2 proteins of influenza virus A.

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    Ly Le

    Full Text Available M2 channel, an influenza virus transmembrane protein, serves as an important target for antiviral drug design. There are still discordances concerning the role of some residues involved in proton transfer as well as the mechanism of inhibition by commercial drugs. The viral M2 proteins show high conservativity; about 3/4 of the positions are occupied by one residue in over 95%. Nine M2 proteins from the H3N2 strain and possibly two proteins from H2N2 strains make a phylogenic cluster closely related to 2RLF. The variability range is limited to 4 residues/position with one exception. The 2RLF protein stands out by the presence of 2 serines at the positions 19 and 50, which are in most other M2 proteins occupied by cysteines. The study of correlated mutations shows that there are several positions with significant mutational correlation that have not been described so far as functionally important. That there are 5 more residues potentially involved in the M2 mechanism of action. The original software used in this work (Consensus Constructor, SSSSg, Corm, Talana is freely accessible as stand-alone offline applications upon request to the authors. The other software used in this work is freely available online for noncommercial purposes at public services on bioinformatics such as ExPASy or NCBI. The study on mutational variability, evolutionary relationship, and correlated mutation presented in this paper is a potential way to explain more completely the role of significant factors in proton channel action and to clarify the inhibition mechanism by specific drugs.

  15. 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.

  16. Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake.

    Science.gov (United States)

    Grinter, Rhys; Josts, Inokentijs; Zeth, Kornelius; Roszak, Aleksander W; McCaughey, Laura C; Cogdell, Richard J; Milner, Joel J; Kelly, Sharon M; Byron, Olwyn; Walker, Daniel

    2014-07-01

    The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.

  17. Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

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    Gwerder Myriam

    2009-12-01

    Full Text Available Abstract Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.

  18. Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes.

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    Noor Haliza Hasan

    Full Text Available A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e protein of avian influenza virus (AIV as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb and chicken antibodies (cAbs recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.

  19. Viral M2 ion channel protein: a promising target for anti-influenza drug discovery.

    Science.gov (United States)

    Moorthy, N S Hari Narayana; Poongavanam, Vasanthanathan; Pratheepa, V

    2014-01-01

    Influenza virus is an important RNA virus causing pandemics (Spanish Flu (1918), Asian Flu (1957), Hong Kong Flu (1968) and Swine Flu (2009)) over the last decades. Due to the spontaneous mutations of these viral proteins, currently available antiviral and anti-influenza drugs quickly develop resistance. To account this, only limited antiinfluenza drugs have been approved for the therapeutic use. These include amantadine and rimantadine (M2 proton channel blockers), zanamivir, oseltamivir and peramivir (neuraminidase inhibitors), favipravir (polymerase inhibitor) and laninamivir. This review provides an outline on the strategies to develop novel, potent chemotherapeutic agents against M2 proton channel. Primarily, the M2 proton channel blockers elicit pharmacological activity through destabilizing the helices by blocking the proton transport across the transmembrane. The biologically important compounds discovered using the scaffolds such as bisnoradmantane, noradamantane, triazine, spiroadamantane, isoxazole, amino alcohol, azaspiro, spirene, pinanamine, etc are reported to exhibit anti-influenza activity against wild or mutant type (S31N and V27A) of M2 proton channel protein. The reported studies explained that the adamantane based compounds (amantadine and rimantadine) strongly interact with His37 (through hydrogen bonding) and Ala30, Ile33 and Gly34 residues (hydrophobic interactions). The adamantane and the non-adamantane scaffolds fit perfectly in the active site pocket present in the wild type and the charged amino groups (ammonium) create positive electrostatic potential, which blocks the transport of protons across the pore. In the mutated proteins, larger or smaller binding pocket are created by small or large mutant residues, which do not allow the molecules fit in the active site. This causes the channel to be unblocked and the protons are allowed to transfer inside the pore. The structural analysis of the M2 proton channel blockers illustrated that

  20. Characterization of M2X formed during 5 MeV Fe2+ irradiation

    Science.gov (United States)

    Getto, E.; Sun, K.; Was, G. S.

    2017-03-01

    The growth of M2X phase in HT9 irradiated to high dpa was explored using self-ion irradiation. HT9 was pre-implanted with 10 appm He and irradiated with a raster-scanned Fe2+ beam with a damage rate of ∼1 × 10-3 dpa/s at 460 °C. The precipitation of M2X was observed and a combination of high resolution transmission electron microscopy (HRTEM), energy filtered transmission electron microscopy (EFTEM) and diffraction analysis was used to characterize the Cr-rich carbide observed at 250 dpa and above. Cr2C was determined to be semi-coherent with the matrix such that [ 100 ] Cr2 C / /[100]α and [ 001 ] Cr2 C / /[ 10 1 bar ] α .with a = 2.71 Å and c = 2.82 Å.

  1. Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus.

    Science.gov (United States)

    Acharya, Rudresh; Carnevale, Vincenzo; Fiorin, Giacomo; Levine, Benjamin G; Polishchuk, Alexei L; Balannik, Victoria; Samish, Ilan; Lamb, Robert A; Pinto, Lawrence H; DeGrado, William F; Klein, Michael L

    2010-08-24

    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(+) and 3(+) with a pK(a) near 6. A 1.65 A 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.

  2. 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.

  3. M5-brane in three-form flux and multiple M2-branes

    CERN Document Server

    Ho, Pei-Ming; Matsuo, Yutaka; Shiba, Shotaro

    2008-01-01

    We investigate the Bagger-Lambert-Gustavsson model associated with the Nambu-Poisson algebra as a theory describing a single M5-brane. We argue that the model is a gauge theory associated with the volume-preserving diffeomorphism in the three-dimenisonal internal space. We derive gauge transformations, actions, supersymmetry transformations, and equations of motions in terms of six-dimensional fields. The equations of motions are written in gauge-covariant form, and the equations for tensor fields have manifest self-dual structure. We demonstrate that the double dimensional reduction of the model reproduces the non-commutative U(1) gauge theory on a D4-brane with a small non-commutativity parameter. We establish relations between parameters in the BLG model and those in M-theory. This shows that the model describes an M5-brane in a large C-field background.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Simulations of the Pore Structures for a M2GlyR Derived Channel Forming Peptide in Different Membrane Environments

    Science.gov (United States)

    Al-Rawi, A.; Herrera, A.; Tomich, J.; Rahman, T.

    2007-03-01

    As part of an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to grasp the characteristics of ion channeling peptides. In particular we present the observed changes in the functional characteristics of NK4-M2GlyR p22 (KKKKPARVGLGITTVLTMTTQS), a M2 GlyR derived channel forming peptide. Starting with a structure determined by multidimensional NMR (800 MHz) in SDS, a potential from CHARMM force-field was used to relax the structure of NK4-M2GlyR p22. Following the relaxation, numerous pore structures were generated for the symmetric five-helix assembly with geometries varying from cylindrical to conical. As it is difficult a priori to assign accurately the orientation of the hydrophilic portion of M2GlyR derived amphipath towards the inside of the pore, we tilted and rotated the helical structure by five different angles about the backbone axis before forming the pore. Energy minimization of the channel was performed in vacuum, in phosphotidylcholine (POPC) membrane, and 60% POPC 30% phosphotidylethanolamine (POPE) in order to determine the effect of the environment surrounding on the structure on its energy minimization. We will present the various pore assemblies, in the different membrane environments, used to predict the most probably membrane bound structure.

  6. Proteins: Form and function

    OpenAIRE

    Roy D Sleator

    2012-01-01

    An overwhelming array of structural variants has evolved from a comparatively small number of protein structural domains; which has in turn facilitated an expanse of functional derivatives. Herein, I review the primary mechanisms which have contributed to the vastness of our existing, and expanding, protein repertoires. Protein function prediction strategies, both sequence and structure based, are also discussed and their associated strengths and weaknesses assessed.

  7. Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

    Science.gov (United States)

    Tsuga, H; Kameyama, K; Haga, T; Honma, T; Lameh, J; Sadée, W

    1998-02-27

    Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

  8. Simulations of the pore structures for a M2G1yR derived channel forming peptide in membrane

    Science.gov (United States)

    Al-Rawi, Ahlam N.; Al-Rawi, Asma; Chen, Jianhan; Herrera, Alvaro; Tomich, John; Rahman, Talat S.

    2008-03-01

    In an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to capture many of the biophysical properties of an intact ion channel using short channel-forming peptides. We have developed two anion selective channel-forming peptides with near native and altered properties from the peptides derived from the glycine receptor: NK4-M2GlyR-p22 WT (KKKKPAR-VGLGITTVLTMTTQS) and NK4-M2GlyR-p22 S22W (KKKKPARVGLGITTVLTMTTQW), respectively. Starting with the two structures determined by solution multidimensional NMR (800 MHz) in SDS, we used CHARMM and NAMD to perform molecular dynamics simulations on the monomers. Using the existing experimental data, we then built an initial 5- helix assembly by altering the tilted angle, rotational angle and pore radius. We investigated the impact of the single mutation at position 22 on the structure and dynamics of the pore formed in a membrane build in a hydrated POPC lipid bilayer. Probable structures for both assemblies are presented.

  9. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    OpenAIRE

    Jacoby, D B; Gleich, G J; Fryer, A. D.

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of ...

  10. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    Science.gov (United States)

    Mirzaei, Nima; Mokhtari Azad, Talat; Nategh, Rakhshandeh; Soleimanjahi, Hoorieh; Amirmozafari, Nour

    2014-01-01

    Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies. PMID:24719728

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

    Energy Technology Data Exchange (ETDEWEB)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon, E-mail: choe@salk.edu [Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (United States)

    2006-09-01

    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{sup +} 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.

  12. Affinity Pull-Down of Proteins Using Anti-FLAG M2 Agarose Beads

    Science.gov (United States)

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    FLAG is an affinity tag widely used for rapid and highly specific one-step protein purification. Native elution of protein from anti-FLAG antibody resins allows the identification of protein and nucleic acid binding partners and functional analysis using biochemical activity assays. PMID:26096505

  13. Na~+/K~+-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication

    Institute of Scientific and Technical Information of China (English)

    GAO; George; Fu

    2010-01-01

    Interplay between the host and influenza virus has a pivotal role for the outcome of infection.The matrix proteins M2/BM2 from influenza (A and B) viruses are small type III integral membrane proteins with a single transmembrane domain,a short amino-terminal ectodomain and a long carboxy-terminal cytoplasmic domain.They function as proton channels,mainly forming a membrane-spanning pore through the transmembrane domain tetramer,and are essential for virus assembly and release of the viral genetic materials in the endosomal fusion process.However,little is known about the host factors which interact with M2/BM2 proteins and the functions of the long cytoplasmic domain are currently unknown.Starting with yeast two-hybrid screening and applying a series of experiments we identified that the β1 subunit of the host Na+/K+-ATPase β1 subunit (ATP1B1) interacts with the cytoplasmic domain of both the M2 and BM2 proteins.A stable ATP1B1 knockdown MDCK cell line was established and we showed that the ATP1B1 knockdown suppressed influenza virus A/WSN/33 replication,implying that the interaction is crucial for influenza virus replication in the host cell.We propose that influenza virus M2/BM2 cytoplasmic domain has an important role in the virus-host interplay and facilitates virus replication.

  14. Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis

    Science.gov (United States)

    Raphael, Itay; Mahesula, Swetha; Purkar, Anjali; Black, David; Catala, Alexis; Gelfond, Jonathon A. L.; Forsthuber, Thomas G.; Haskins, William E.

    2014-09-01

    Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

  15. Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals.

    Science.gov (United States)

    Gao, Xueliang; Wang, Haizhen; Yang, Jenny J; Chen, Jing; Jie, Jiang; Li, Liangwei; Zhang, Yinwei; Liu, Zhi-Ren

    2013-05-31

    Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

  16. 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.

  17. 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)

  18. Prokaryote-expressed M2e protein improves H9N2 influenza vaccine efficacy and protection against lethal influenza A virus in mice

    National Research Council Canada - National Science Library

    Kim, Eun-Ha; Lee, Jun-Han; Pascua, Philippe Noriel Q; Song, Min-Suk; Baek, Yun-Hee; Kwon, Hyeok-Il; Park, Su-Jin; Lim, Gyo-Jin; Decano, Arun; Chowdhury, Mohammed Ye; Seo, Su-Kyung; Song, Man Ki; Kim, Chul-Joong; Choi, Young-Ki

    2013-01-01

    .... For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2...

  19. 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.

  20. Association of m1 and m2 muscarinic receptor proteins with asymmetric synapses in the primate cerebral cortex: morphological evidence for cholinergic modulation of excitatory neurotransmission.

    OpenAIRE

    Mrzljak, L; Levey, A I; Goldman-Rakic, P S

    1993-01-01

    Muscarinic m1 receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant m1 and m2 muscarinic receptor proteins, we have demonstrated that both m1 and m2 receptors are prominently ...

  1. 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

  2. Recombinant M2e protein-based ELISA: a novel and inexpensive approach for differentiating avian influenza infected chickens from vaccinated ones.

    Directory of Open Access Journals (Sweden)

    Farhid Hemmatzadeh

    Full Text Available Available avian influenza (AIV serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e of avian influenza virus (H5N1 strain was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.

  3. 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.

  4. Asscessment of the clinical value of CK-M2 and oligosaccharide protein inserum from patients with gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Gui Lan Zhao; Jie Lin; Shu You Peng; Qi Yun Li; Fan Zhou; Xian Sheng Chen; Xin You Xie; Yi Xiong Zheng

    2000-01-01

    AIM To evaluate the clinical value of creatine kinase macroisoenzyme type 2 (CK-M2) and oligosaccharideprotein (OP) in serum from patients with gastric carcinoma (GC).METHODS Serum level of CK-M2 was detected by agar gel electrophoresis. OP concentration was measuredby an enzyme immunoassay.RESULTS Serum levels of CK-M2 and OP in 57 cases of GC were significantly higher than those in 51 caseswith gastric precancerous lesion and 28 controls. The diagnostic sensitivity and specificity for GC with CK-M2 was 56.10% and 98.63% respectively. CK-M2 and OP were not associated with histologic type and degreeof differentiation.CONCLUSION These results suggest that CK-M2 may serve as a marker to diagnose GC, and the specificityis higher, whereas OP is not more significant for GC diagnosis, but it could be a useful indicator forevaluation the status of body immune.

  5. Murid Gammaherpesvirus Latency-Associated Protein M2 Promotes the Formation of Conjugates between Transformed B Lymphoma Cells and T Helper Cells.

    Directory of Open Access Journals (Sweden)

    Diana Fontinha

    Full Text Available Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4 depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

  6. Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals.

    Science.gov (United States)

    Minaba, Masaomi; Ichiyama, Susumu; Kojima, Katsura; Ozaki, Mamiko; Kato, Yusuke

    2006-12-01

    Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, G alpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian G alpha(i/o)-coupled receptors. GOA-1 and M2 were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5'-[3-O-thio]triphosphate ([35S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M2-G alpha(i/o) coupling. These results indicate that the M2 activates GOA-1 as well as G alpha(i/o).

  7. 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

    1999-01-01

    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 ten

  8. 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

  9. Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant.

    Science.gov (United States)

    Zhao, Guangyu; Du, Lanying; Xiao, Wenjun; Sun, Shihui; Lin, Yongping; Chen, Min; Kou, Zhihua; He, Yuxian; Lustigman, Sara; Jiang, Shibo; Zheng, Bo-Jian; Zhou, Yusen

    2010-10-18

    Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Down-regulation of muscarinic acetylcholine receptor M2 adversely affects the expression of Alzheimer's disease-relevant genes and proteins.

    Science.gov (United States)

    Zuchner, Thole; Schliebs, Reinhard; Perez-Polo, J Regino

    2005-10-01

    Beta-amyloid peptides play a major role in the pathogenesis of Alzheimer's disease (AD). Therefore, preventing beta-amyloid formation by inhibition of the beta site amyloid precursor protein-cleaving enzyme (BACE) 1 is considered as a potential strategy to treat AD. Cholinergic mechanisms have been shown to control amyloid precursor protein processing and the number of muscarinic M2-acetylcholine receptors is decreased in brain regions of patients with AD enriched with senile plaques. Therefore, the present study investigates the effect of this M2 muscarinic receptor down-regulation by siRNA on total gene expression and on regulation of BACE1 in particular in SK-SH-SY5Y cells. This model system was used for microarray analysis after carbachol stimulation of siRNA-treated cells compared with carbachol stimulated, non-siRNA-treated cells. The same model system was used to elucidate changes at the protein level by using two-dimensional gels followed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) analysis. Taken together, the results indicate that the M2 acetylcholine receptor down-regulation in brains of patients with AD has important effects on the expression of several genes and proteins with major functions in the pathology of AD. This includes beta-secretase BACE1 as well as several modulators of the tau protein and other AD-relevant genes and proteins. Moreover, most of these genes and proteins are adversely affected against the background of AD.

  11. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    Science.gov (United States)

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

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

    Science.gov (United States)

    Rangaswamy, Udaya S; Speck, Samuel H

    2014-01-01

    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.

  13. 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.

  14. Minimal Integral Submanifolds of Sasakian Space Form%Sasaki空间形式( ̄M)2n+1(c)中极小积分子流形

    Institute of Scientific and Technical Information of China (English)

    周亚非

    2005-01-01

    设( ̄M)2n+1(c)是2n+1维常φ-截面曲率c的 Sasaki 空间形式,Mn是( ̄M)2n+1(c)(c>-3)的n维紧致极小积分子流形、 S. Maeda(Tensor N S,1981,35:200~204.) 证明了:当n≥5时,若M的 Ricci 曲率满足Ric(M n)>(n-2-1/n)·c+3/4,则M n是全测地的.讨论了n=4的情形,得到类似的结果.

  15. Homogeneous M2 duals

    CERN Document Server

    Figueroa-O'Farrill, José

    2015-01-01

    Motivated by the search for new gravity duals to M2 branes with $N>4$ supersymmetry --- equivalently, M-theory backgrounds with Killing superalgebra $\\mathfrak{osp}(N|4)$ for $N>4$ --- we classify (except for a small gap) homogeneous M-theory backgrounds with symmetry Lie algebra $\\mathfrak{so}(n) \\oplus \\mathfrak{so}(3,2)$ 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 $\\operatorname{AdS}_4 \\times P^7$, with $P$ riemannian and homogeneous under the action of $\\operatorname{SO}(5)$, or $S^4 \\times Q^7$ with $Q$ lorentzian and homogeneous under the action of $\\operatorname{SO}(3,2)$. 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.

  16. The M2 Channel

    DEFF Research Database (Denmark)

    Santner, Paul

    and inhibition mechanisms, drug design studies were recently able to achieve successes in finding new potent inhibitors, some of which are even able to inhibit resistant M2 variants. Effective and robust methods for measuring M2 activity on the other hand are still scarce and tactics to assess the genetic...... barrier of new inhibitors as well as resistance development non-existent. Therefore we developed a fluorescence sensor based assay that directly measures proton conduction (pHlux assay) and combined it with an already established directed evolution selection and screening system of M2 to identify possible...... 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....

  17. In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803.

    Science.gov (United States)

    Bourrelle-Langlois, Maxime; Morrow, Geneviève; Finet, Stéphanie; Tanguay, Robert M

    2016-01-01

    We previously reported the in silico characterization of Synechococcus sp. phage 18 kDa small heat shock protein (HspSP-ShM2). This small heat shock protein (sHSP) contains a highly conserved core alpha crystalline domain of 92 amino acids and relatively short N- and C-terminal arms, the later containing the classical C-terminal anchoring module motif (L-X-I/L/V). Here we establish the oligomeric profile of HspSP-ShM2 and its structural dynamics under in vitro experimental conditions using size exclusion chromatography (SEC/FPLC), gradient native gels electrophoresis and dynamic light scattering (DLS). Under native conditions, HspSP-ShM2 displays the ability to form large oligomers and shows a polydisperse profile. At higher temperatures, it shows extensive structural dynamics and undergoes conformational changes through an increased of subunit rearrangement and formation of sub-oligomeric species. We also demonstrate its capacity to prevent the aggregation of citrate synthase, malate dehydrogenase and luciferase under heat shock conditions through the formation of stable and soluble hetero-oligomeric complexes (sHSP:substrate). In contrast, the host cyanobacteria Synechococcus sp. WH7803 15 kDa sHSP (HspS-WH7803) aggregates when in the same conditions as HspSP-ShM2. However, its solubility can be maintained in the presence of non-ionic detergent Triton™X-100 and forms an oligomeric structure estimated to be between dimer and tetramer but exhibits no apparent inducible structural dynamics neither chaperon-like activity in all the assays and molar ratios tested. SEC/FPLC and thermal aggregation prevention assays results indicate no formation of hetero-oligomeric complex or functional interactions between both sHSPs. Taken together these in vitro results portray the phage HspSP-ShM2 as a classical sHSP and suggest that it may be functional at the in vivo level while behaving differently than its host amphitropic sHSP.

  18. Effects of beta-amyloid protein on M1 and M2 subtypes of muscarinic acetylcholine receptors in the medial septum-diagonal band complex of the rat: relationship with cholinergic, GABAergic, and calcium-binding protein perikarya.

    Science.gov (United States)

    González, Iván; Arévalo-Serrano, Juan; Sanz-Anquela, José Miguel; Gonzalo-Ruiz, Alicia

    2007-06-01

    Cortical cholinergic dysfunction has been correlated with the expression and processing of beta-amyloid precursor protein. However, it remains unclear as to how cholinergic dysfunction and beta-amyloid (Abeta) formation and deposition might be related to one another. Since the M1- and M2 subtypes of muscarinic acetylcholine receptors (mAChRs) are considered key molecules that transduce the cholinergic message, the purpose of the present study was to assess the effects of the injected Abeta peptide on the number of M1mAchR- and M2mAChR-immunoreactive cells in the medial septum-diagonal band (MS-nDBB) complex of the rat. Injections of Abeta protein into the retrosplenial cortex resulted in a decrease in M1mAChR and M2mAChR immunoreactivity in the MS-nDBB complex. Quantitative analysis revealed a significant reduction in the number of M1mAChR- and M2mAChR-immunoreactive cells in the medial septum nucleus (MS) and in the horizontal nucleus of the diagonal band of Broca (HDB) as compared to the corresponding hemisphere in control animals and with that seen in the contralateral hemisphere, which corresponds to the PBS-injected side. Co-localization studies showed that the M1mAChR protein is localized in GABA-immunoreactive cells of the MS-nDBB complex, in particular those of the MS nucleus, while M2mAChR protein is localized in both the cholinergic and GABAergic cells. Moreover, GABAergic cells containing M2mAChR are mainly localized in the MS nucleus, while cholinergic cells containing M2mAChR are localized in the MS and the HDB nuclei. Our findings suggest that Abeta induces a reduction in M1mAChR- and M2mAChR-containing cells, which may contribute to impairments of cholinergic and GABAergic transmission in the MS-nDBB complex.

  19. 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.

  20. Analytical applications for pore-forming proteins.

    Science.gov (United States)

    Kasianowicz, John J; Balijepalli, Arvind K; Ettedgui, Jessica; Forstater, Jacob H; Wang, Haiyan; Zhang, Huisheng; Robertson, Joseph W F

    2016-03-01

    Proteinaceous nanometer-scale pores are ubiquitous in biology. The canonical ionic channels (e.g., those that transport Na(+), K(+), Ca(2+), and Cl(-) across cell membranes) play key roles in many cellular processes, including nerve and muscle activity. Another class of channels includes bacterial pore-forming toxins, which disrupt cell function, and can lead to cell death. We describe here the recent development of these toxins for a wide range of biological sensing applications. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  1. Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State NMR of Oriented Bicelles

    Science.gov (United States)

    Wang, Tuo; Hong, Mei

    2015-01-01

    A wide variety of membrane proteins induce membrane curvature for function, thus it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state NMR methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static 31P NMR spectra of magnetically oriented DMPC/DHPC bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the peptide with the ability to generate a high-curvature phase. 2D 31P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. 31P- and 13C-detected 1H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the 31P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. Based on this resolution, 2D 1H-31P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not the DHPC 31P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the M2 amphipathic helix induces high membrane curvature and localizes the protein to this phase, in excellent agreement with the membrane-scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are

  2. Effect of Nanosize SiO2 Particles Added into Electrolyte on the Composition and Morphology of Oxide Layers Formed in Alloy AK6M2 Under Microarc Oxidizing

    Science.gov (United States)

    Krishtal, M. M.; Ivashin, P. V.; Yasnikov, I. S.; Polunin, A. V.

    2015-11-01

    Oxide layers formed on AK6M2 aluminum-silicon alloy by microarc oxidizing (MO) are studied. The chemical and phase compositions and the morphology of the layers deposited from a base-composition electrolyte with different contents of added SiO2 powder are determined. It is shown that high-temperature phases form in the oxide layer at a specific concentration of SiO2 nanoparticles. This indicates elevation of the effective temperature in the zone of synthesis of the oxide layers in the process of MO. The addition of nanosize particles of SiO2 into the electrolyte influences positively the operating characteristics of the formed oxide layer.

  3. Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.

    Science.gov (United States)

    Wang, Tuo; Hong, Mei

    2015-04-07

    A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static (31)P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D) (31)P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. (31)P- and (13)C-detected (1)H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the (31)P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D (1)H-(31)P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not DHPC (31)P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good

  4. Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

    Science.gov (United States)

    Hayashi, M K; Haga, T

    1997-04-15

    A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

  5. Highly viscous dough-forming properties of marama protein.

    Science.gov (United States)

    Amonsou, Eric O; Taylor, John R N; Naushad Emmambux, M; Gyebi Duodu, K; Minnaar, Amanda

    2012-10-01

    Marama bean is an indigenous southern African oilseed legume with an unusual protein composition. Hence, its rheological properties were studied. Marama protein formed a highly viscous and extensible dough when compared to soya and gluten. With a dough of 38% moisture, marama protein extensibility was very high (304% of its original length), twice that of gluten and soya, and this increased considerably (>3-fold) when the moisture content was increased to 45%. With added peroxidase, the storage modulus (G') of marama protein dough increased with time, suggesting the formation of new and strong protein networks. Dityrosine crosslinks were detected in the doughs. Marama protein showed a single transition with a denaturation temperature higher than soya glycinin. Marama protein was more hydrophobic and contained more β-sheet structure than did soya. Thus, the highly viscous and extensible rheological behaviour of marama protein is probably related to its high β-sheet conformation, hydrophobic interactions and tyrosine crosslinks.

  6. Folding of Aggregated Proteins to Functionally Active Form

    Science.gov (United States)

    2006-06-01

    in Cn5 were reformed with a total protein yield of 87% and 100% recovery of activity [34]. Artificial chaperone-assisted refolding This method...typically formed using a mixture of reduced/oxidizedCurrent Opinion in Biotechnology 2006, 17:367–372 370 Protein technologiesglutathione, and glycerol

  7. Maternal low protein diet leads to placental angiogenic compensation via dysregulated M1/M2 macrophages and TNFa expression in Sprague-Dawley rats

    Science.gov (United States)

    A maternal low-protein (LP) diet results in low birth weight, increased offspring rapid adipose tissue catch-up growth, adult obesity, and insulin resistance in Sprague-Dawley rats. The placenta plays key roles in nutrient transport and fetal growth. Placental function is dependent on regulation of ...

  8. 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.

  9. The SARS coronavirus nucleocapsid protein--forms and functions.

    Science.gov (United States)

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Tachyonic Anti-M2 Branes

    CERN Document Server

    Bena, Iosif; Kuperstein, Stanislav; Massai, Stefano

    2014-01-01

    We study the dynamics of anti-M2 branes in a warped Stenzel solution with M2 charges dissolved in fluxes by taking into account their full backreaction on the geometry. The resulting supergravity solution has a singular magnetic four-form flux in the near-brane region. We examine the possible resolution of this singularity via the polarization of anti-M2 branes into M5 branes, and compute the corresponding polarization potential for branes smeared on the finite-size four-sphere at the tip of the Stenzel space. We find that the potential has no minimum. We then use the potential for smeared branes to compute the one corresponding to a stack of localized anti-M2 branes, and use this potential to compute the force between two anti-M2 branes at tip of the Stenzel space. We find that this force, which is zero in the probe approximation, is in fact repulsive. This surprising result points to a tachyonic instability of anti-M2 branes in backgrounds with M2 brane charge dissolved in flux.

  11. Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2.

    Science.gov (United States)

    Tsuga, H; Kameyama, K; Haga, T; Kurose, H; Nagao, T

    1994-12-23

    Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.

  12. Anti-influenza M2e antibody

    Science.gov (United States)

    Bradbury, Andrew M.

    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.

  13. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

    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.

  14. Anti-influenza M2e antibody

    Science.gov (United States)

    Bradbury, Andrew M.

    2013-04-16

    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.

  15. 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.

  16. The open-close mechanism of M2 channel protein in influenza A virus: A computational study on the hydrogen bonds and cation-π interactions among His37 and Trp41

    Institute of Scientific and Technical Information of China (English)

    CHENG JiaGao; ZHU WeiLiang; WANG YanLi; YAN XiuHua; LI Zhong; TANG Yun; JIANG HuaLiang

    2008-01-01

    The M2 protein from influenza A virus is a tetrameric ion channel. It was reported that the permeation of the ion channel is correlated with the hydrogen bond network among His37 residues and the cation-π interactions between His37 and Trp41. In the present study, the hydrogen bonding network of 4-methyl-imidazoles was built to mimic the hydrogen bonds between His37 residues, and the cation-π interactions between 4-methyl-imidazolium and indole systems were selected to represent the interac-tions between His37 and Trp41. Then, quantum chemistry calculations at the MP2/6-311G** level were carried out to explore the properties of the hydrogen bonds and the cation-π interactions. The calculation results indicate that the binding strength of the N--H…N hydrogen bond between imidazole rings is up to -6.22 kcal·mol-1, and the binding strength of the strongest cation-π interaction is up to -18.8 kcal·mol-1 (T-shaped interaction) or -12.3 kcal·mol-1 (parallel stacking interaction). Thus, the calculated binding energies indicate that it is possible to control the permeation of the M2 ion channel through the hydrogen bond network and the cation-π interactions by altering the pH values.

  17. The open-close mechanism of M2 channel protein in influenza A virus:A computational study on the hydrogen bonds and cation-π interactions among His37 and Trp41

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The M2 protein from influenza A virus is a tetrameric ion channel. It was reported that the permeation of the ion channel is correlated with the hydrogen bond network among His37 residues and the cation-π interactions between His37 and Trp41. In the present study,the hydrogen bonding network of 4-methyl-imidazoles was built to mimic the hydrogen bonds between His37 residues,and the cation-π interactions between 4-methyl-imidazolium and indole systems were selected to represent the interac-tions between His37 and Trp41. Then,quantum chemistry calculations at the MP2/6-311G level were carried out to explore the properties of the hydrogen bonds and the cation-π interactions. The calcula-tion results indicate that the binding strength of the N-H···N hydrogen bond between imidazole rings is up to -6.22 kcal·mol-1,and the binding strength of the strongest cation-π interaction is up to -18.8 kcal·mol-1(T-shaped interaction) or -12.3 kcal·mol-1(parallel stacking interaction). Thus,the calcu-lated binding energies indicate that it is possible to control the permeation of the M2 ion channel through the hydrogen bond network and the cation-π interactions by altering the pH values.

  18. Long-lived reactive species formed on proteins induce changes in protein and lipid turnover.

    Science.gov (United States)

    Davies, Michael

    2014-10-01

    Proteins are major targets for oxidative damage in vivo due to their high abundance and rapid rates of reaction with both one-electron (radical) and two-electron oxidants (e.g. singlet oxygen, hypochlorous acid, peroxynitrous acid, reactive aldehydes). The turnover of both native and modified proteins is critical for maintenance of cell homeostasis, with this occurring via multiple pathways including proteasomes (for cytosolic species), the Lon protease (in mitochondria), and the endo-lysosomal systems (both extra- and intra-cellular species). Evidence has been presented for both enhanced and diminished rates of catabolism of modified proteins, as well as altered turnover of native (unmodified) proteins as a result of damage to these systems, potentially as a result of the accumulation of damaged proteins. In recent studies we have shown that long-lived reactive species forms on proteins (hydroperoxides, chloramines and aldehydes) can modify the activity of proteasomal and lysosomal enzymes. Some of the above species are efficient inhibitors of the tryptic and chymotryptic activities of the 26S proteasome, as well as lysosomal cathepsin and acid lipase activities. These are key species in the turnover of both proteins and lipoproteins. The loss of enzyme activity is accompanied in many cases, by oxidation of critical thiol residues via molecular reactions. For reactive aldehydes (either free or protein-bound) direct enzyme inhibition can occur as well as modulation of protein levels and, in the case of lysosomes, changes in lysosomal numbers. Overall, these data indicate that the formation of reactive species on proteins can modulate cell function by multiple pathways including interference with the turnover of native proteins (including critical cell signalling molecules) and alterations in the rate of clearance of modified proteins. Both pathways may contribute to the development of a number of human pathologies associated with oxidative damage.

  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. 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

    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-assemble in combinat...

  1. 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...

  2. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    Science.gov (United States)

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  3. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    OpenAIRE

    Wymke Ockenga; Ritva Tikkanen

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimul...

  4. 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.

  5. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    Science.gov (United States)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

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

    Science.gov (United States)

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn 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

    2014-06-19

    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.

  7. 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.

  8. 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...

  9. Periodic Arrays of M2-Branes

    CERN Document Server

    Jeon, Imtak; Richmond, Paul

    2012-01-01

    We consider periodic arrays of M2-branes in the ABJM model in the spirit of a circle compactification to D2-branes in type IIA string theory. The result is a curious formulation of three-dimensional maximally supersymmetric Yang-Mills theory in terms of fermions, seven transverse scalars, a non-dynamical gauge field and an additional scalar `dual gluon'. Upon further T-duality on a transverse torus we obtain a non-manifest-Lorentz-invariant description of five-dimensional maximally supersymmetric Yang-Mills. Here the additional scalar field can be thought of as the components of a two-form along the torus. This action can be viewed as an M-theory description of M5-branes on ${\\mathbb T}^3$.

  10. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya (Stanford-MED); (Kyoto); (Gakushuin); (Kyushu)

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  11. Evidence of paired M2 muscarinic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.; Ferrendelli, C.A.; Fisher, A.; Hanchett, H.E.; Zhang, R. (Univ. of Miami School of Medicine, FL (USA))

    1991-02-01

    Binding assays involving various antagonists, including N-(3H) methylscopolamine, (3H)quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat brainstem (medulla, pons, and colliculi). However, competition curves between N-(3H)methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of (3H)-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with (3H)quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.

  12. What is $\\Delta m^2_{ee}$ ?

    CERN Document Server

    Parke, Stephen

    2016-01-01

    The current short baseline reactor experiments, Daya Bay and RENO (Double Chooz) have measured (or are capable of measuring) an effective $\\Delta m^2$ associated with the atmospheric oscillation scale of 0.5 km/MeV in electron anti-neutrino disappearance. In this paper, I compare and contrast the different definitions of such an effective $\\Delta m^2$ and argue that the simple, L/E independent, definition given by $\\Delta m^2_{ee} \\equiv \\cos^2 \\theta_{12} \\Delta m^2_{31}+ \\sin^2 \\theta_{12} \\Delta m^2_{32}$, i.e. "the $\

  13. Electrophoretic separation method for membrane pore-forming proteins in multilayer lipid membranes.

    Science.gov (United States)

    Okamoto, Yukihiro; Tsujimoto, Yusuke; Umakoshi, Hiroshi

    2016-03-01

    In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore-forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high-performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore-forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore-forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore-forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high-performance by utilizing the specific properties of lipid membranes.

  14. M2 factor of four-petal Gaussian beam

    Institute of Scientific and Technical Information of China (English)

    Zhou Guo-Quan; Fan Yan

    2008-01-01

    Based on the second-order moments,this paper derives an analytical expression of the M2 factor of four-petal Gaussian beam.The results show that the M2 factor is only determined by the beam order n.The corresponding numerical calculations are also given.As the beam order increases,the augment of M2 factor is disciplinary.As the expression of M2 factor is expressed in series form and becomes more complicated,a new concise formula of M2 factor is also presented by using curve fitting of numerical calculations.When 3≤n≤200,the maximum error rate of fitting formula will not exceed 2.6% and the average error rate is 0.28%.This research is helpful to the applications of four-petal Gaussian beam.

  15. Vaccination with recombinant 4 × M2e.HSP70c fusion protein as a universal vaccine candidate enhances both humoral and cell-mediated immune responses and decreases viral shedding against experimental challenge of H9N2 influenza in chickens.

    Science.gov (United States)

    Dabaghian, Mehran; Latify, Ali Mohammad; Tebianian, Majid; Nili, Hassan; Ranjbar, Ali Reza Tevangar; Mirjalili, Ali; Mohammadi, Mashallah; Banihashemi, Reza; Ebrahimi, Seyyed Mahmoud

    2014-11-07

    As cellular immunity is essential for virus clearance, it is commonly accepted that no adequate cellular immunity is achieved by all available inactivated HA-based influenza vaccines. Thus, an improved influenza vaccine to induce both humoral and cell-mediated immune responses is urgently required to control LPAI H9N2 outbreaks in poultry farms. M2e-based vaccines have been suggested and developed as a new generation of universal vaccine candidate against influenza A infection. Our previous study have shown that a prime-boost administration of recombinant 4×M2e.HSP70c (r4M2e/H70c) fusion protein compared to conventional HA-based influenza vaccines provided full protection against lethal dose of influenza A viruses in mice. In the present study, the immunogenicity and protective efficacy of (r4M2e/H70c) was examined in chickens. The data reported herein show that protection against H9N2 viral challenge was significantly increased in chickens by injection of r4M2e/H70c compared with injection of conventional HA-based influenza vaccine adjuvanted with MF59 or recombinant 4×M2e (r4M2e) without HSP70c. Oropharyngeal and cloacal shedding of the virus was detected in all of the r4M2e/H70c vaccinated birds at 2 days after challenge, but the titer was low and decreased rapidly to reach undetectable levels at 7 days after challenge. Moreover, comparison of protective efficacy against LPAI H9N2 in birds intramuscularly immunized with r4M2e/H70c likely represented the ability of the M2e-based vaccine in providing cross-protection against heterosubtypic H9N2 challenge and also allowed the host immune system to induce HA-homosubtype neutralizing antibody against H9N2 challenge. This protective immunity might be attributed to enhanced cell-mediated immunity, which is interpreted as increased lymphocytes proliferation, increased levels of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines production and increased CD4(+) to CD8(+) ratios, resulting from the injection of four tandem

  16. 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 nature. The

  17. Controlling the self-assembly of protein polymers via heterodimer-forming modules

    NARCIS (Netherlands)

    Domeradzka, Natalia Eliza

    2016-01-01

    Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanost

  18. M2M massive wireless access

    DEFF Research Database (Denmark)

    Zanella, Andrea; Zorzi, Michele; Santos, André F.

    2013-01-01

    of the current cellular standards. Here, we provide insights and introduce potential solutions for the cellular radio protocol that will allow the efficient support of Machine-to-Machine (M2M) communications. The paper focuses on the massive aspect of M2M. We will introduce PHY and MAC approaches such as Coded...... and research guidelines for enabling future networks to support efficiently M2M communications....

  19. 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

    2017-01-01

    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. PMID:28176876

  20. 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.

  1. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.

    OpenAIRE

    1984-01-01

    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  2. Purification in a functional form of the terminal protein of Bacillus subtilis phage ø29

    OpenAIRE

    1984-01-01

    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  3. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    Science.gov (United States)

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  4. Hofbauer cells of M2a, M2b and M2c polarization may regulate feto-placental angiogenesis.

    Science.gov (United States)

    Loegl, J; Hiden, U; Nussbaumer, E; Schliefsteiner, C; Cvitic, S; Lang, I; Wadsack, C; Huppertz, B; Desoye, G

    2016-11-01

    The human placenta comprises a special type of tissue macrophages, the Hofbauer cells (HBC), which exhibit M2 macrophage phenotype. Several subtypes of M2-polarized macrophages (M2a, M2b and M2c) exist in almost all tissues. Macrophage polarization depends on the way of macrophage activation and leads to the expression of specific cell surface markers and the acquisition of specific functions, including tissue remodeling and the promotion of angiogenesis. The placenta is a highly vascularized and rapidly growing organ, suggesting a role of HBC in feto-placental angiogenesis. We here aimed to characterize the specific polarization and phenotype of HBC and investigated the role of HBC in feto-placental angiogenesis. Therefore, HBC were isolated from third trimester placentas and their phenotype was determined by the presence of cell surface markers (FACS analysis) and secretion of cytokines (ELISA). HBC conditioned medium (CM) was analyzed for pro-angiogenic factors, and the effect of HBC CM on angiogenesis, proliferation and chemoattraction of isolated primary feto-placental endothelial cells (fpEC) was determined in vitro Our results revealed that isolated HBC possess an M2 polarization, with M2a, M2b and M2c characteristics. HBC secreted the pro-angiogenic molecules VEGF and FGF2. Furthermore, HBC CM stimulated the in vitro angiogenesis of fpEC. However, compared with control medium, chemoattraction of fpEC toward HBC CM was reduced. Proliferation of fpEC was not affected by HBC CM. These findings demonstrate a paracrine regulation of feto-placental angiogenesis by HBC in vitro Based on our collective results, we propose that the changes in HBC number or phenotype may affect feto-placental angiogenesis. © 2016 Society for Reproduction and Fertility.

  5. Assigning error to an M2 measurement

    Science.gov (United States)

    Ross, T. Sean

    2006-02-01

    The ISO 11146:1999 standard has been published for 6 years and set forth the proper way to measure the M2 parameter. In spite of the strong experimental guidance given by this standard and the many commercial devices based upon ISO 11146, it is still the custom to quote M2 measurements without any reference to significant figures or error estimation. To the author's knowledge, no commercial M2 measurement device includes error estimation. There exists, perhaps, a false belief that M2 numbers are high precision and of insignificant error. This paradigm causes program managers and purchasers to over-specify a beam quality parameter and researchers not to question the accuracy and precision of their M2 measurements. This paper will examine the experimental sources of error in an M2 measurement including discretization error, CCD noise, discrete filter sets, noise equivalent aperture estimation, laser fluctuation and curve fitting error. These sources of error will be explained in their experimental context and convenient formula given to properly estimate error in a given M2 measurement. This work is the result of the author's inability to find error estimation and disclosure of methods in commercial beam quality measurement devices and building an ISO 11146 compliant, computer- automated M2 measurement device and the resulting lessons learned and concepts developed.

  6. TNF counterbalances the emergence of M2 tumor macrophages

    Science.gov (United States)

    Kratochvill, Franz; Neale, Geoffrey; Haverkamp, Jessica M.; de Velde, Lee-Ann Van; Smith, Amber M.; Kawauchi, Daisuke; McEvoy, Justina; Roussel, Martine F.; Dyer, Michael A.; Qualls, Joseph E.; Murray, Peter J.

    2015-01-01

    Cancer is a form of 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. PMID:26365184

  7. JNK3 phosphorylates Bax protein and induces ability to form pore on bilayer lipid membrane

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Bax is a pro-apoptotic cytosolic protein. In this work native (unphosphorylated and JNK3 phosphorylated Bax proteins are studied on artificial bilayer membranes for pore formation. Phosphorylated Bax formed pore on the bilayer lipid membrane whereas native one does not. In cells undergoing apoptosis the pore formed by the phosphorylated Bax could be important in cytochrome c release from the mitochondrial intermembrane space to the cytosol. The low conductance (1.5 nS of the open state of the phosphorylated Bax pore corresponds to pore diameter of 0.9 nm which is small to release cytochrome c (∼3.4 nm. We hypothesized that JNK3 phosphorylated Bax protein can form bigger pores after forming complexes with other mitochondrial proteins like VDAC, t-Bid etc. to release cytochrome c.

  8. Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

    Science.gov (United States)

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  9. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Wymke Ockenga

    2015-05-01

    Full Text Available The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  10. 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 an

  11. Characterization of the aggregates formed during recombinant protein expression in bacteria

    OpenAIRE

    de Marco Ario; Schrödel Andrea

    2005-01-01

    Abstract Background The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates. Results The total lysate separated into 4 different fractions whereas only the one with the lowest density wa...

  12. Characterization of M2 antibodies in asymptomatic Chinese population

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hua Jiang; Ren-Qian Zhong; Xiao-Yun Fan; Yin Hu; Feng An; Jian-Wen Sun; Xian-Tao Kong

    2003-01-01

    AIM: To investigate the presence of M2 antibodies specific for pdmary biliary cirrhosis (PBC) in asymptomatic Chinese and identify patients with early PBC.METHODS: Enzyme-linked immunosorbent assay (ElISA)tests for M2 antibodies to recombinant protein were performed in 5 011 subjects (age range, 26-85 years; mean age: 45.81±15.02 years) who took an annual physical examination. M2-positive subjects were further analyzed for immunoglobulin (Ig) classes and subclasses of M2 antibodies.Clinical, biochemical and immunological data were obtained for M2-positive subjects. In addition, ultrasonography (US)or endoscopic retrograde cholangio-pancreatography (ERCP)was performed to exclude any disorders other than PBC.RESULTS: M2 antibodies were detected in 8 (0.16%) of the 5 0LL subjects studied. Of the 8 subjects, 7 were female and 1 was male (age range: 40-74 years). An unexplained increase of serum alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (γ-GT) values, often to striking levels,was detected in 4 M2-positive subjects, 3 of them accorded with the diagnostic criteria recommended by the American Association for the Study of Liver Diseases, even though they had no symptoms of PBC (such as fatigue, pruritus or jaundice).Liver biopsy was performed in two M2-positive subjects and the histology was compatible with PBC in both cases.CONCLUSION: Our data, while not assessing the true prevalence of asymptomatic PBC in the general population,suggest that asymptomatic PBC is much more common in China than has been supposed.

  13. Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy

    Directory of Open Access Journals (Sweden)

    Katarzyna Popiolek-Barczyk

    2015-01-01

    Full Text Available Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL on the chronic constriction injury to the sciatic nerve (CCI-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t. was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker but did not change GFAP (an astrocyte marker on day 7 after CCI. PTL reduced the protein level of M1 (IL-1β, IL-18, and iNOS and enhanced M2 (IL-10, TIMP1 factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1β, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes.

  14. Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy.

    Science.gov (United States)

    Popiolek-Barczyk, Katarzyna; Kolosowska, Natalia; Piotrowska, Anna; Makuch, Wioletta; Rojewska, Ewelina; Jurga, Agnieszka M; Pilat, Dominika; Mika, Joanna

    2015-01-01

    Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1β, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1β, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes.

  15. Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy

    Science.gov (United States)

    Popiolek-Barczyk, Katarzyna; Kolosowska, Natalia; Makuch, Wioletta; Rojewska, Ewelina; Jurga, Agnieszka M.; Pilat, Dominika

    2015-01-01

    Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1β, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1β, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes. PMID:26090236

  16. Purification and characterization of coacervate-forming cuticular proteins from Papilio xuthus pupae.

    Science.gov (United States)

    Yamanaka, Masahiro; Ishizaki, Yumi; Nakagawa, Taro; Taoka, Azuma; Fukumori, Yoshihiro

    2013-07-01

    The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming. Transmission electron microscopy analysis revealed that these proteins assemble ordered bead-like ultrastructures. We cloned and sequenced PX-1 and PX-4 cDNAs. The PX-1 and PX-4 amino acid sequences contain many hydrophobic residues and show homologies to insect cuticular proteins. Moreover, when recombinant PX-1 and PX-4 were overexpressed in Escherichia coli, both recombinant proteins exhibited temperature-dependent coacervation. Furthermore, analyses of truncated mutants of PX-1 suggest that both the Val/Pro-rich region and Gly/lle-rich regions of PX-1 are involved in such coacervation.

  17. Potential recombinant vaccine against influenza A virus based on M2e displayed on nodaviral capsid nanoparticles

    Directory of Open Access Journals (Sweden)

    Yong CY

    2015-04-01

    Full Text Available Chean Yeah Yong,1 Swee Keong Yeap,2 Kok Lian Ho,3 Abdul Rahman Omar,2,4 Wen Siang Tan1,2 1Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Bioscience, 3Department of Pathology, Faculty of Medicine and Health Sciences, 4Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia Abstract: Influenza A virus poses a major threat to human health, causing outbreaks from time to time. Currently available vaccines employ inactivated viruses of different strains to provide protection against influenza virus infection. However, high mutation rates of influenza virus hemagglutinin (H and neuraminidase (N glycoproteins give rise to vaccine escape mutants. Thus, an effective vaccine providing protection against all strains of influenza virus would be a valuable asset. The ectodomain of matrix 2 protein (M2e was found to be highly conserved despite mutations of the H and N glycoproteins. Hence, one to five copies of M2e were fused to the carboxyl-terminal end of the recombinant nodavirus capsid protein derived from Macrobrachium rosenbergii. The chimeric proteins harboring up to five copies of M2e formed nanosized virus-like particles approximately 30 nm in diameter, which could be purified easily by immobilized metal affinity chromatography. BALB/c mice immunized subcutaneously with these chimeric proteins developed antibodies specifically against M2e, and the titer was proportional to the copy numbers of M2e displayed on the nodavirus capsid nanoparticles. The fusion proteins also induced a type 1 T helper immune response. Collectively, M2e displayed on the nodavirus capsid nanoparticles could provide an alternative solution to a possible influenza pandemic in the future. Keywords: matrix 2 ectodomain, nodavirus capsid, virus-like particle, fusion protein, subunit vaccine, immunogenicity

  18. Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

    Science.gov (United States)

    Zhao, Guangyu; Miao, Yu; Guo, Yan; Qiu, Hongjie; Sun, Shihui; Kou, Zhihua; Yu, Hong; Li, Junfeng; Chen, Yue; Jiang, Shibo; Du, Lanying; Zhou, Yusen

    2014-01-01

    Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

  19. M2M massive wireless access

    DEFF Research Database (Denmark)

    Zanella, Andrea; Zorzi, Michele; Santos, André F.

    2013-01-01

    In order to make the Internet of Things a reality, ubiquitous coverage and low-complexity connectivity are required. Cellular networks are hence the most straightforward and realistic solution to enable a massive deployment of always connected Machines around the globe. Nevertheless, a paradigm...... of the current cellular standards. Here, we provide insights and introduce potential solutions for the cellular radio protocol that will allow the efficient support of Machine-to-Machine (M2M) communications. The paper focuses on the massive aspect of M2M. We will introduce PHY and MAC approaches such as Coded...

  20. The identification of foam-forming soluble proteins from wheat (Triticum aestivum) dough.

    Science.gov (United States)

    Salt, Louise J; Robertson, James A; Jenkins, John A; Mulholland, Francis; Mills, E N Clare

    2005-04-01

    Proteomic methods have been used to identify foam-forming soluble proteins from dough that may play an important role in stabilising gas bubbles in dough, and hence influence the crumb structure of bread. Proteins from a soluble fraction of dough (dough liquor) or dough liquor foam have been separated by two-dimensional gel electrophoresis, and 42 identified using a combination of matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight analyses. Major polypeptide components included beta-amylase, tritin and serpins, with members of the alpha-amylase/trypsin inhibitor family being particularly abundant. Neither prolamin seed storage proteins nor the surface-active protein puroindoline were found. Commonly used dough ingredients (NaCl, Na L-ascorbate) had only a minor effect on the 2-DE protein profiles of dough liquor, of which one of the more significant was the loss of 9 kDa nonspecific lipid transfer protein. Many proteins were lost in dough liquor foam, particularly tritin, whilst a number of alpha-amylase inhibitors were more dominant, suggesting that these are amongst the most strongly surface-active proteins in dough liquor. Such proteins may play a role determining the ability of the aqueous phase of doughs, as represented by dough liquor, to form an elastic interface lining the bubbles, and hence maintain their integrity during dough proving.

  1. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

    Science.gov (United States)

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-10-18

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  2. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    Directory of Open Access Journals (Sweden)

    Jacek Dygut

    2016-10-01

    Full Text Available The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1 Shared hydrophobic cores (spanning the whole dimer; (2 Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  3. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.

    Science.gov (United States)

    Prieto, I; Lázaro, J M; García, J A; Hermoso, J M; Salas, M

    1984-03-01

    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3.

  4. Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells.

    Science.gov (United States)

    Kryndushkin, Dmitry; Pripuzova, Natalia; Burnett, Barrington G; Shewmaker, Frank

    2013-09-20

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

  5. Computational methods in preformulation study for pharmaceutical solid dosage forms of therapeutic proteins

    Science.gov (United States)

    Majee, Sutapa Biswas; Biswas, Gopa Roy

    2017-06-01

    Design and delivery of protein-based biopharmaceuticals needs detailed planning and strict monitoring of intermediate processing steps, storage conditions and container-closure system to ensure a stable, elegant and biopharmaceutically acceptable dosage form. Selection of manufacturing process variables and conditions along with packaging specifications can be achieved through properly designed preformulation study protocol for the formulation. Thermodynamic stability and biological activity of therapeutic proteins depend on folding-unfolding and three-dimensional packing dynamics of amino acid network in the protein molecule. Lack of favourable environment may cause protein aggregation with loss in activity and even fatal immunological reaction. Although lyophilization can enhance the stability of protein-based formulations in the solid state, it can induce protein unfolding leading to thermodynamic instability. Formulation stabilizers such as preservatives can also result in aggregation of therapeutic proteins. Modern instrumental techniques in conjunction with computational tools enable rapid and accurate prediction of amino acid sequence, thermodynamic parameters associated with protein folding and detection of aggregation "hot-spots." Globular proteins pose a challenge during investigations on their aggregation propensity. Biobetter therapeutic monoclonal antibodies with enhanced stability, solubility and reduced immunogenic potential can be designed through mutation of aggregation-prone zones. The objective of the present review article is to focus on the various analytical methods and computational approaches used in the study of thermodynamic stability and aggregation tendency of therapeutic proteins, with an aim to develop optimal and marketable formulation. Knowledge of protein dynamics through application of computational tools will provide the essential inputs and relevant information for successful and meaningful completion of preformulation studies on

  6. Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

    Science.gov (United States)

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

    2016-05-01

    Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

  7. Cell surface expression of glycosylated, nonglycosylated, and truncated forms of a cytoplasmic protein pyruvate kinase.

    Science.gov (United States)

    Hiebert, S W; Lamb, R A

    1988-09-01

    The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80% of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible that specific signals may not be needed in the ectodomain of this hybrid protein to specify cell surface localization.

  8. RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes.

    Science.gov (United States)

    Foldi, Julia; Shang, Yingli; Zhao, Baohong; Ivashkiv, Lionel B; Hu, Xiaoyu

    2016-03-01

    Development of alternatively activated (M2) macrophage phenotypes is a complex process that is coordinately regulated by a plethora of pathways and factors. Here, we report that RBP-J, a DNA-binding protein that integrates signals from multiple pathways including the Notch pathway, is critically involved in polarization of M2 macrophages. Mice deficient in RBP-J in the myeloid compartment exhibited impaired M2 phenotypes in vivo in a chitin-induced model of M2 polarization. Consistent with the in vivo findings, M2 polarization was partially compromised in vitro in Rbpj-deficient macrophages as demonstrated by reduced expression of a subset of M2 effector molecules including arginase 1. Functionally, myeloid Rbpj deficiency impaired M2 effector functions including recruitment of eosinophils and suppression of T cell proliferation. Collectively, we have identified RBP-J as an essential regulator of differentiation and function of alternatively activated macrophages.

  9. 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.

  10. Activation and dynamic network of the M2 muscarinic receptor

    OpenAIRE

    Miao, Yinglong; Nichols, Sara E.; Gasper, Paul M.; Metzger, Vincent T; McCammon, J. Andrew

    2013-01-01

    G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist...

  11. The Metastasis-associated Proteins 1 and 2 Form Distinct Protein Complexes with Histone Deacetylase Activity

    Institute of Scientific and Technical Information of China (English)

    Ya-LiYao; Wcn-MingYang

    2005-01-01

    The metastasis-associated protein MTA1 has been shown to express differentially to high levels in metastatic cells. MTA2, which is homologous to MTA1, is a component of the NURD ATP-dependcnt chromatin remodeling and histone deacetylase complex. Here we report evidence that although both human MTA1 and MTA2 repress transcription specifically, are located in the nucleus, and contain associated histone deacetylase activity, they exist in two biochemically distinct protein complexes and may perform different functions pertaining to tumor metastasis. Specifically, both MTA1 and MTA2 complexes exert histone deacetylase activity. However, the MTA1 complex contained HDAC1/2, RbAp46/48, and MBD3, but not Sin3 or Mi2, two important components of the MTA2 complex. Moreover, the MTA2 complex is similar to the HDAC1 complex, suggesting a housekeeping role of the MTA2 complex. The MTA1 complex could be further separated, resulting in acore MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity were integral properties of the MTA1 complex. Finally, MTA1, unlike MTA2, did not interact with the pleotropic transcription factor YY1 or the immunophilin FKBP25. We suggest that MTA1 associates with adifferent set of transcription factors from MTA2 and that this property may contribute to the metastatic potential of cells overexpressing MTA1. We also report the finding of human MTA3, which is highly homologous toboth MTA1 and MTA2. However, MTA3 does not repress transcription to a significant level and appears to have a diffused pattern of subcellular localization, suggesting a biological role distinct from that of the other two MTA proteins.

  12. Superconformal indices and M2-branes

    Energy Technology Data Exchange (ETDEWEB)

    Eager, Richard [Kavli Institute for the Physics and Mathematics of the Universe (WPI),University of Tokyo, Kashiwa, Chiba 277-8583 (Japan); Schmude, Johannes [RIKEN Nishina Center, Saitama 351-0198 (Japan)

    2015-12-10

    We derive the superconformal index of the world-volume theory on M2-branes probing the cone over an arbitrary Sasaki-Einstein seven-manifold. The index is expressed in terms of the cohomology groups of the cone. We match our supergravity results with known results from gauge theory. Along the way we derive the spectrum of short Kaluza-Klein multiplets on generic Sasaki-Einstein seven-manifolds.

  13. Superconformal indices and M2-branes

    Science.gov (United States)

    Eager, Richard; Schmude, Johannes

    2015-12-01

    We derive the superconformal index of the world-volume theory on M2-branes probing the cone over an arbitrary Sasaki-Einstein seven-manifold. The index is expressed in terms of the cohomology groups of the cone. We match our supergravity results with known results from gauge theory. Along the way we derive the spectrum of short Kaluza-Klein multiplets on generic Sasaki-Einstein seven-manifolds.

  14. Soluble Proteins Form Film by the Treatment of Low Temperature Plasma

    Science.gov (United States)

    Ikehara, Sanae; Sakakita, Hajime; Ishikawa, Kenji; Akimoto, Yoshihiro; Nakanishi, Hayao; Shimizu, Nobuyuki; Hori, Masaru; Ikehara, Yuzuru

    2015-09-01

    It has been pointed out that low temperature plasma in atmosphere was feasible to use for hemostasis without heat injury. Indeed, earlier studies demonstrated that low temperature plasma played an important role to stimulate platelets to aggregate and turned on the proteolytic activities of coagulation factors, resulting in the acceleration of the natural blood coagulation process. On the other hands, our developed equips could immediately form clots upon the contact with plasma flair, while the histological appearance was different from natural coagulation. Based on these findings in formed clots, we sought to determine if plasma flair supplied by our devices was capable of forming film using a series of soluble proteins Following plasma treatment, films were formed from bovine serum albumin, and the other plasma proteins at physiological concentration. Analysis of trans-electron microscope demonstrated that plasma treatment generated small protein particles and made them fuse to be larger aggregations The combined results demonstrated that plasma are capable of aggregating soluble proteins and that platelets and coagulation factors are not necessary for plasma induced blood coagulation. Supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.).

  15. Dielectrophoretic manipulation and solubility of protein nanofibrils formed from crude crystallins

    DEFF Research Database (Denmark)

    Domigan, Laura; Andersen, Karsten B.; Sasso, Luigi

    2013-01-01

    Protein nanofibrils and nanotubes are now widely accepted as having potential for use in the field of bionanotechnology. For this to be a feasible alternative to existing technologies, there is a need for a commercially viable source. Previous work has identified amyloid fibrils formed from crude...

  16. M2e-Based Universal Influenza A Vaccines

    Directory of Open Access Journals (Sweden)

    Lei Deng

    2015-02-01

    Full Text Available The successful isolation of a human influenza virus in 1933 was soon followed by the first attempts to develop an influenza vaccine. Nowadays, vaccination is still the most effective method to prevent human influenza disease. However, licensed influenza vaccines offer protection against antigenically matching viruses, and the composition of these vaccines needs to be updated nearly every year. Vaccines that target conserved epitopes of influenza viruses would in principle not require such updating and would probably have a considerable positive impact on global human health in case of a pandemic outbreak. The extracellular domain of Matrix 2 (M2e protein is an evolutionarily conserved region in influenza A viruses and a promising epitope for designing a universal influenza vaccine. Here we review the seminal and recent studies that focused on M2e as a vaccine antigen. We address the mechanism of action and the clinical development of M2e-vaccines. Finally, we try to foresee how M2e-based vaccines could be implemented clinically in the future.

  17. Pore-Forming Proteins as Mediators of Novel Epigenetic Mechanism of Epilepsy

    Science.gov (United States)

    Surguchov, Andrei; Surgucheva, Irina; Sharma, Mukut; Sharma, Ram; Singh, Vikas

    2017-01-01

    Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures. In the last two decades, numerous gene defects underlying different forms of epilepsy have been identified with most of these genes encoding ion channel proteins. Despite these developments, the etiology of majority of non-familial epilepsies has no known associated genetic mutations and cannot be explained by defects in identified ion channels alone. We hypothesize that de novo formation of ion channels by naturally unfolded proteins (NUPs) increases neuronal excitability. Altered ionic homeostasis may initiate/contribute to cellular cascades related to epileptogenesis in susceptible individuals. Here, we consider two small proteins, namely, α-synuclein and stefin B, as prototypical candidates to illustrate the underlying mechanism(s). Previous work points to an association between epilepsy and α-synuclein or stefin B, but the mechanism(s) underlying such association remains elusive. We review the evidence to link the structure–function of these proteins with disease processes. Epigenetic mechanisms unrelated to altered DNA sequence(s) that may affect epileptogenesis include transcriptional or posttranscriptional regulation. Such epigenetic mechanisms or their combination(s) enhance the levels of these proteins and as a result the ability to form annular structures, which upon incorporation into membrane form novel ion channels and disturb intracellular ion homeostasis. Alternative epigenetic mechanisms may change amyloidogenic proteins by posttranslational modifications, thereby increasing their propensity to form channels. Further research elucidating the details about the formation of ion channels through these mechanisms and their role in epileptogenesis may define new molecular targets and guide the development of new drug targets.

  18. Functional analysis of stress protein data in a flor yeast subjected to a biofilm forming condition

    Directory of Open Access Journals (Sweden)

    Jaime Moreno-García

    2016-06-01

    Full Text Available In this data article, an OFFGEL fractionator coupled to LTQ Orbitrap XL MS equipment and a SGD filtering were used to detect in a biofilm-forming flor yeast strain, the maximum possible number of stress proteins under the first stage of a biofilm formation conditions (BFC and under an initial stage of fermentation used as reference, so-called non-biofilm formation condition (NBFC. Protein functional analysis – based on cellular components and biological process GO terms – was performed for these proteins through the SGD Gene Ontology Slim Mapper tool. A detailed analysis and interpretation of the data can be found in “Stress responsive proteins of a flor yeast strain during the early stages of biofilm formation” [1].

  19. Functional analysis of stress protein data in a flor yeast subjected to a biofilm forming condition

    Science.gov (United States)

    Moreno-García, Jaime; Mauricio, Juan Carlos; Moreno, Juan; García-Martínez, Teresa

    2016-01-01

    In this data article, an OFFGEL fractionator coupled to LTQ Orbitrap XL MS equipment and a SGD filtering were used to detect in a biofilm-forming flor yeast strain, the maximum possible number of stress proteins under the first stage of a biofilm formation conditions (BFC) and under an initial stage of fermentation used as reference, so-called non-biofilm formation condition (NBFC). Protein functional analysis – based on cellular components and biological process GO terms – was performed for these proteins through the SGD Gene Ontology Slim Mapper tool. A detailed analysis and interpretation of the data can be found in “Stress responsive proteins of a flor yeast strain during the early stages of biofilm formation” [1]. PMID:27104213

  20. Presynaptic muscarinic M(2) receptors modulate glutamatergic transmission in the bed nucleus of the stria terminalis.

    Science.gov (United States)

    Guo, Ji-Dong; Hazra, Rimi; Dabrowska, Joanna; Muly, E Chris; Wess, Jürgen; Rainnie, Donald G

    2012-03-01

    The anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) serves as an important relay station in stress circuitry. Limbic inputs to the BNST(ALG) are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNST(ALG). Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNST(ALG). Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analog, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired-pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M(2) receptor-preferring antagonists, and was absent in M(2)/M(4) receptor knockout mice, suggesting that presynaptic M(2) receptors mediate the effect of ACh. Immunoelectron microscopy studies further revealed the presence of M(2) receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M(2) receptors might be an important modulator of the stress circuit and hence a novel target for drug development.

  1. Secondary and quaternary structures of the (+)-pinoresinol-forming dirigent protein.

    Science.gov (United States)

    Halls, Steven C; Lewis, Norman G

    2002-07-30

    The (+)-pinoresinol-forming dirigent protein is the first protein capable of stereoselectively coupling two coniferyl alcohol derived radical species, in this case to give the 8-8' linked (+)-pinoresinol. Only dimeric cross-linked dirigent protein structures were isolated when 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide was used as cross-linking agent, whereas the associated oxidase, presumed to generate the corresponding free radical substrate, was not detected. Native Forsythia intermedia dirigent protein isoforms were additionally subjected to MALDI-TOF and ESI-MS analyses, which established the presence of both monomeric masses of 23-25 kDa and dimeric dirigent protein species ranging from 46 to 49 kDa. Analytical ultracentrifugation, sedimentation velocity, and sedimentation equilibrium analyses of the native dirigent protein in open solution confirmed further its dimeric nature as well as a propensity to aggregate, with the latter being dependent upon both temperature and solution ionic strength. Circular dichroism analysis suggested that the dirigent protein was primarily composed of beta-sheet and loop structures.

  2. Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation.

    Science.gov (United States)

    Salem-Izacc, S M; Jesuino, R S; Brito, W A; Pereira, M; Felipe, M S; Soares, C M

    1997-01-01

    In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

  3. Casein kinase Ⅱ interacts with prion protein in vitro and forms complex with na-tive prion protein in vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase Ⅱ (CK2) is a ubiquitously expressed and evolutiouarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2α and CK2β complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2α and glutathione S-transferase-histidine-CK2β were expressed in Escherichia coll. The interaction between CK2 and PrP was evaluated with co-immunoprecipi-tation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2α, but not with CK2β. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2α subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2α subunit. The domain responsible for interacting with CK2α was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2α (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.

  4. 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.

  5. LOSA-M2 aerosol Raman lidar

    Energy Technology Data Exchange (ETDEWEB)

    Balin, Yu S; Bairashin, G S; Kokhanenko, G P; Penner, I E; Samoilova, S V [V.E. Zuev Institute of Atmospheric Optics, Siberian Branch, Russian Academy of Sciences, Tomsk (Russian Federation)

    2011-10-31

    The scanning LOSA-M2 aerosol Raman lidar, which is aimed at probing atmosphere at wavelengths of 532 and 1064 nm, is described. The backscattered light is received simultaneously in two regimes: analogue and photon-counting. Along with the signals of elastic light scattering at the initial wavelengths, a 607-nm Raman signal from molecular nitrogen is also recorded. It is shown that the height range of atmosphere probing can be expanded from the near-Earth layer to stratosphere using two (near- and far-field) receiving telescopes, and analogue and photon-counting lidar signals can be combined into one signal. Examples of natural measurements of aerosol stratification in atmosphere along vertical and horizontal paths during the expeditions to the Gobi Desert (Mongolia) and Lake Baikal areas are presented.

  6. The (178m2)Hf Controversy

    Energy Technology Data Exchange (ETDEWEB)

    Becker, J A; Gemmell, D S; Schiffer, J P; Wilhelmy, J B

    2003-07-24

    Since its discovery in the 1960's the {sup 178m2}Hf isomer has garnered high attention from both the basic and applied communities in nuclear science. It's combination of high spin (16+), long half life (31 yrs), and high excitation energy (2.446 MeV) offer unique possibilities as an energy storage medium. Interest in the isomer was rekindled beginning in 1999 when a series of publications began to appear from a group (referred to here as the ''Texas collaboration'') primarily based at the University of Texas, Dallas [1]. They reported observations that some of the stored energy could be released (''triggered'') when the isomer was exposed to a fluence of photons in the energy range {approx}10 to {approx}60 keV. The implications of this observation are profound. Even though the claimed cross section for the process was {approx}7 orders of magnitude greater than would be predicted from the known systematics of photon absorption by nuclei in this mass range [2], such a highly efficient method for triggering the isomeric deexcitation immediately suggested applications utilizing the explosive or the controlled gradual energy release from a very compact source. The prospect of such applications has focused considerable interest on realizing the promise that is implicit in the reported observations. However, two experiments performed by a group from ANL/LANL/LLNL at the Advanced Photon Source at Argonne (the ''APS collaboration'') reported negative results for the observation of any photon-triggered deexcitation of the {sup 178m2}Hf isomer [3]. This has led to a continued controversy, where both sides have adamantly defended their observations. At this point an outsider has difficulty determining whether there is indeed a triggering effect that should be pursued energetically with substantial resources, or whether the phenomenon consists of overly optimistic interpretation of data.

  7. Mode of action of antimicrobial proteins, pore-forming toxins and biologically active

    Directory of Open Access Journals (Sweden)

    O Schmidt

    2005-07-01

    Full Text Available Antimicrobial peptides and pore-forming toxins are important effectors in innate immune defencereactions. But their mode of action, comprising the insertion into cholesterol-containing membranes isnot known. Here we explore the mechanical implications of pore-formation by extracellular proteinassemblies that drive cellular uptake reactions by leverage-mediated (LM processes, whereoligomeric adhesion molecules bent membrane-receptors around ‘hinge’-like lipophorin particles. Theinteractions of antimicrobial peptides, pore-forming toxins and biologically active proteins with LMassembliesprovide a new paradigm for the configurational specificity and sterical selectivity ofbiologically active peptides.

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

    OpenAIRE

    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...

  9. Bax and Bif-1 proteins interact on Bilayer Lipid Membrane and form pore.

    Science.gov (United States)

    Gupta, Rajeev; Ghosh, Subhendu

    2015-08-07

    Bax and Bax interacting factor-1(Bif-1) are cytosolic proteins, which translocate towards mitochondria during mitochondria-mediated apoptosis. Bif-1 has been identified to co-immunoprecipitate with Bax in apoptotic cells. We have studied the interaction of Bax and Bif-1 on Bilayer Lipid Membrane (BLM) through electrophysiological experiments. It has been observed that Bax-Bif-1 equimolar mixture can form a pore. The pore conductance is in the range of 4.96-5.41 nS. It also displays a sub-state with a conductance of 2.6 nS. No pore activity is observed on BLM when monomeric Bax and Bif-1 proteins are tested independently. The above-mentioned pore forming activity could be relevant in mitochondria-mediated apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

    Science.gov (United States)

    Bullard, Steven A; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C; Dierdorff, Jason M; Ebert, Scott M; DeLau, Austin D; Gibson, Bradford W; Adams, Christopher M

    2016-08-19

    Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. 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...... on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology....

  12. The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat.

    Science.gov (United States)

    Suzuki, Hiroshi; Ito, Yasuyuki; Yamazaki, Yuji; Mineta, Katsuhiko; Uji, Masami; Abe, Kazuhiro; Tani, Kazutoshi; Fujiyoshi, Yoshinori; Tsukita, Sachiko

    2013-01-01

    Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

  13. Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols.

    Science.gov (United States)

    Kadoya, Saori; Fujii, Kahori; Izutsu, Ken-ichi; Yonemochi, Etsuo; Terada, Katsuhide; Yomota, Chikako; Kawanishi, Toru

    2010-04-15

    Physical properties and protein-stabilizing effects of sugar alcohols in frozen aqueous solutions and freeze-dried solids were studied. Various frozen sugar alcohol solutions showed a glass transition of the maximally freeze-concentrated phase at temperatures (T(g)'s) that depended largely on the solute molecular weights. Some oligosaccharide-derived sugar alcohols (e.g., maltitol, lactitol, maltotriitol) formed glass-state amorphous cake-structure freeze-dried solids. Microscopic observation of frozen maltitol and lactitol solutions under vacuum (FDM) indicated onset of physical collapse at temperatures (T(c)) several degrees higher than their T(g)'s. Freeze-drying of pentitols (e.g., xylitol) and hexitols (e.g., sorbitol, mannitol) resulted in collapsed or crystallized solids. The glass-forming sugar alcohols prevented activity loss of a model protein (LDH: lactate dehydrogenase) during freeze-drying and subsequent storage at 50 degrees C. They also protected bovine serum albumin (BSA) from lyophilization-induced secondary structure perturbation. The glass-forming sugar alcohols showed lower susceptibility to Maillard reaction with co-lyophilized L-lysine compared to reducing and non-reducing disaccharides during storage at elevated temperature. Application of the oligosaccharide-derived sugar alcohols as alternative stabilizers in lyophilized protein formulations was discussed. Copyright 2010 Elsevier B.V. All rights reserved.

  14. The Promyelocytic Leukemia Gene Product (PML) Forms Stable Complexes with the Retinoblastoma Protein

    Science.gov (United States)

    Alcalay, Myriam; Tomassoni, Lucia; Colombo, Emanuela; Stoldt, Stephan; Grignani, Francesco; Fagioli, Marta; Szekely, Laszlo; Helin, Kristian; Pelicci, Pier Giuseppe

    1998-01-01

    PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor α (RARα) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RARα. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RARα expression. Both PML and PML-RARα form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RARα involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RARα, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RARα further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RARα protein may derive from the alteration of PML-regulated transcription. PMID:9448006

  15. Growth of Helicobacter pylori in a long spiral form does not alter expression of immunodominant proteins

    Directory of Open Access Journals (Sweden)

    Proujansky Roy

    2002-09-01

    Full Text Available Abstract Background We have previously reported that altered culture conditions (a broth media with shaking could induce a strain of Helicobacter pylori to assume a long spiral morphology resembling that described for Helicobacter heilmannii. The present study was initiated to determine if other strains of H. pylori could be induced to assume that morphology and if doing so would alter the expression of immunodominant proteins. Results The six strains used in this study were American Type Culture Collection 43504, 43579, 49503, 51652, and 51653, and Sydney Strain I. Each strain was grown on solid media and in broth culture using conditions previously shown to induce the long spiral morphology in strain 43504. DNA from each was subjected to urease gene fingerprint analysis. Results of the molecular analysis showed identical fingerprint patterns for each strain independent of culture source, indicating that only a single strain was present in each culture. Expression of immunodominant proteins was assessed by SDS polyacrylamide gel electrophoresis and Western blotting with hyperimmune rabbit anti H. pylori sera or serum from an H. pylori infected patient. Analysis of protein profiles revealed some variation between strains but no significant differences associated with morphologic alterations. Conclusions These results indicate that growth of H. pylori in a long spiral form does not affect expression of immunodominant proteins, thus in vivo growth in the long spiral form (not documented to date would not be distinguishable by serology.

  16. 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.

  17. 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.

  18. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    Science.gov (United States)

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  19. Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome.

    Directory of Open Access Journals (Sweden)

    Danae Schulz

    2015-12-01

    Full Text Available Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG. In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

  20. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza.

    Directory of Open Access Journals (Sweden)

    Byoung-Shik Shim

    Full Text Available BACKGROUND: The ectodomain of matrix protein 2 (M2e of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n. route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l. route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. METHODS AND RESULTS: A recombinant M2 protein with three tandem copies of the M2e (3M2eC was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. CONCLUSIONS: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

  1. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    Science.gov (United States)

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  2. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    Science.gov (United States)

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

  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. Superconformal M2-branes and generalized Jordan triple systems

    CERN Document Server

    Nilsson, Bengt E W

    2008-01-01

    Three-dimensional conformal theories with six supersymmetries and SU(4) R-symmetry describing stacks of M2-branes are here proposed to be related to generalized Jordan triple systems. Writing the four-index structure constants in an appropriate form, the Chern-Simons part of the action immediately suggests a connection to such triple systems. In this note we show that the whole theory with six manifest supersymmetries can be naturally expressed in terms of structure constants of generalized Jordan triple systems. We comment on the associated graded Lie algebra, which corresponds to an extension of the gauge group.

  5. [Some properties of complexes formed by small heat shock proteins with denatured actin].

    Science.gov (United States)

    Pivovarova, A V; Chebotareva, N A; Guseev, N B; Levitskiĭ, D I

    2008-01-01

    We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.

  6. 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.

  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. Kinetics of Proton Transport into Influenza Virions by the Viral M2 Channel

    NARCIS (Netherlands)

    Ivanovic, Tijana; Rozendaal, Rutger; Floyd, Daniel L.; Popovic, Milos; Oijen, Antoine M. van; Harrison, Stephen C.

    2012-01-01

    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 a

  9. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

    Science.gov (United States)

    Formosa-Dague, Cécile; Speziale, Pietro; Foster, Timothy J; Geoghegan, Joan A; Dufrêne, Yves F

    2016-01-12

    Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

  10. 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; Namba, Shigetou

    2015-01-01

    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. 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 throughout the cell, and we

  11. F-actin-like filaments formed by plasmid segregation protein ParM

    DEFF Research Database (Denmark)

    van den Ent, Fusinita; Møller-Jensen, Jakob; Amos, Linda A.;

    2002-01-01

    It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis...... of Escherichia coli formed by ParM, a plasmid-encoded protein required for accurate segregation of low-copy-number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F-actin) and MreB filaments that maintain the cell shape...

  12. The Role of a Novel Topological Form of the Prion Protein in Prion Disease

    Science.gov (United States)

    2008-07-01

    are characterizing the phenotype of these mice to further our understanding of CtmPrP– meditated neurotoxicity, which we believe will shed new light...Biol 160:529 –539. Hegde RS, Voigt S, Lingappa VR (1998a) Regulation of protein topology by trans-acting factors at the endoplasmic reticulum. Mol...Cell 2:85–91. Hegde RS, Mastrianni JA, Scott MR, Defea KA, Tremblay P, Torchia M, DeArmond SJ, Prusiner SB, Lingappa VR (1998b) A transmembrane form of

  13. Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

    Science.gov (United States)

    Azam, Anum; Li, Cheng; Metcalf, Kevin J; Tullman-Ercek, Danielle

    2016-11-01

    Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its

  14. Toxicity and oxidative stress of different forms of organic selenium and dietary protein in mallard ducklings

    Science.gov (United States)

    Hoffman, D.J.; Heinz, G.H.; LeCaptain, L.J.; Eisemann, J.D.; Pendleton, G.W.

    1996-01-01

    Concentrations of over 100 ppm (mg/kg) selenium (Se) have been found in aquatic plants and insects associated with irrigation drainwater and toxicity to fish and wildlife. Composition of diet for wild ducklings may vary in selenium-contaminated environments. Earlier studies have compared toxicities and oxidative stress of Se as selenite to those of seleno-DL-methionine (DL) in mallards (Anas platyrhynchos). 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 except for 30 ppm Se as W. 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 at 30 ppm in the diet was the most toxic form, resulting in high mortality (64%) and impaired growth (>50%) in survivors and the greatest increase in ratio of oxidized to reduced hepatic glutathione (GSH). Se as both L and DL decreased the concentrations of hepatic GSH and total thiols. Se as Y accumulated the least in liver (approximately 50% of other forms) and had less effect on GSH and total thiols. In a second experiment, in which the basal diet was a commercial duck feed (22 % protein), survival was not affected by 30 ppm Se as DL, L, or Y and oxidative effects on GSH metabolism were less pronounced than with the wheat diet.

  15. Half-BPS M2-brane orbifolds

    CERN Document Server

    de Medeiros, Paul

    2010-01-01

    Smooth (at least 1/2)-BPS Freund-Rubin backgrounds of eleven-dimensional supergravity of the form AdS_4 x X^7 have been recently classified. Requiring that amount of supersymmetry forces X to be a spherical space form, whence isometric to the quotient of the round 7-sphere by a freely-acting finite subgroup of SO(8). The classification is given in terms of ADE subgroups of the quaternions embedded in SO(8) as the graph of an automorphism. In this paper we extend this classification by dropping the requirement that the background be smooth, so that X is now allowed to be an orbifold of the round 7-sphere. We find that if the background is (more than 1/2)-BPS, then it is automatically smooth in accordance with the homogeneity conjecture, but that there are many half-BPS orbifolds, most of them new. The classification is now given in terms of pairs of ADE subgroups of quaternions fibred over the same finite group. We classify such subgroups and then describe the resulting orbifolds in terms of iterated quotients...

  16. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

    Directory of Open Access Journals (Sweden)

    Skrypkina I. Ya.

    2011-06-01

    Full Text Available Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China); Yan Changling [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China); Wang Xuejing [Chemistry and Chemical Engineer School, Henna Institute of Science and Technology, Xinxiang 453003 (China); Wang Gongke [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China)

    2009-12-15

    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{sup 2} g{sup -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. Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones.

    Science.gov (United States)

    Macrander, Jason; Daly, Marymegan

    2016-12-08

    Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved.

  19. Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins in Sea Anemones

    Directory of Open Access Journals (Sweden)

    Jason Macrander

    2016-12-01

    Full Text Available Sea anemones (Cnidaria, Anthozoa, and Actiniaria use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea. Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena; these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC binding site and the N-terminal region involved with cell membrane penetration were highly conserved.

  20. 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.

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

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    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.

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

    Science.gov (United States)

    Donnelly, M. Lauren; Li, William; Li, Yong-qing; Hinkel, Lauren; Setlow, Peter

    2017-01-01

    ABSTRACT 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. PMID:28096487

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

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    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.

  4. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein.

    Science.gov (United States)

    Lecours, Katia; Tremblay, Marie-Hélène; Gagné, Marie-Eve Laliberté; Gagné, Stéphane M; Leclerc, Denis

    2006-05-01

    Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27-215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27-215 was 15N labeled for NMR studies and a 2D 1H-15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27-215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.

  5. Dynamically-expressed prion-like proteins form a cuticle in the pharynx of Caenorhabditis elegans

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    Julia B. George-Raizen

    2014-10-01

    Full Text Available In molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In the nematode Caenorhabditis elegans there are two types of cuticle: a well-studied collagenous cuticle lines the body, and a poorly-understood chitinous cuticle lines the pharynx. In the posterior end of the pharynx is the grinder, a tooth-like cuticular specialization that crushes food prior to transport to the intestine for digestion. We here show that the grinder increases in size only during the molt. To gain molecular insight into the structure of the grinder and pharyngeal cuticle, we performed a microarray analysis to identify mRNAs increased during the molt. We found strong transcriptional induction during the molt of 12 of 15 previously identified abu genes encoding Prion-like (P glutamine (Q and asparagine (N rich PQN proteins, as well as 15 additional genes encoding closely related PQN proteins. abu/pqn genes, which we name the abu/pqn paralog group (APPG genes, were expressed in pharyngeal cells and the proteins encoded by two APPG genes we tested localized to the pharyngeal cuticle. Deleting the APPG gene abu-14 caused abnormal pharyngeal cuticular structures and knocking down other APPG genes resulted in abnormal cuticular function. We propose that APPG proteins promote the assembly and function of a unique cuticular structure. The strong developmental regulation of the APPG genes raises the possibility that such genes would be identified in transcriptional profiling experiments in which the animals' developmental stage is not precisely staged.

  6. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    Science.gov (United States)

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

  7. Brane Tilings, M2-branes and Orbifolds

    CERN Document Server

    Davey, John

    2011-01-01

    Brane Tilings represent one of the largest classes of superconformal theories with known gravity duals in 3+1 and also 2+1 dimensions. They provide a useful link between a large class of quiver gauge theories and their moduli spaces, which are the toric Calabi-Yau (CY) singularities. This thesis includes a discussion of an algorithm that can be used to generate all brane tilings with any given number of superpotential terms. All tilings with at most 8 superpotential terms have been generated using an implementation of this method. Orbifolds are a subject of central importance in string theory. It is widely known that there may be two or more orbifolds of a space by a finite group. Abelian Calabi-Yau orbifolds of the form $\\BC^3 / \\Gamma$ can be counted according to the size of the group $|\\Gamma|$. Three methods of counting these orbifolds will be given. A brane tiling together with a set of Chern Simons levels is sufficient to define a quiver Chern-Simons theory which describes the worldvolume theory of the ...

  8. 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; Barrangou, Rodolphe; Klaenhammer, Todd R

    2015-10-16

    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.

  9. Vaccine safety and efficacy evaluation of a recombinant bovine respiratory syncytial virus (BRSV) with deletion of the SH gene and subunit vaccines based on recombinant human RSV proteins: N-nanorings, P and M2-1, in calves with maternal antibodies.

    Science.gov (United States)

    Blodörn, Krister; Hägglund, Sara; Fix, Jenna; Dubuquoy, Catherine; Makabi-Panzu, Boby; Thom, Michelle; Karlsson, Per; Roque, Jean-Louis; Karlstam, Erika; Pringle, John; Eléouët, Jean-François; Riffault, Sabine; Taylor, Geraldine; Valarcher, Jean François

    2014-01-01

    The development of safe and effective vaccines against both bovine and human respiratory syncytial viruses (BRSV, HRSV) to be used in the presence of RSV-specific maternally-derived antibodies (MDA) remains a high priority in human and veterinary medicine. Herein, we present safety and efficacy results from a virulent BRSV challenge of calves with MDA, which were immunized with one of three vaccine candidates that allow serological differentiation of infected from vaccinated animals (DIVA): an SH gene-deleted recombinant BRSV (ΔSHrBRSV), and two subunit (SU) formulations based on HRSV-P, -M2-1, and -N recombinant proteins displaying BRSV-F and -G epitopes, adjuvanted by either oil emulsion (Montanide ISA71VG, SUMont) or immunostimulating complex matrices (AbISCO-300, SUAbis). Whereas all control animals developed severe respiratory disease and shed high levels of virus following BRSV challenge, ΔSHrBRSV-immunized calves demonstrated almost complete clinical and virological protection five weeks after a single intranasal vaccination. Although mucosal vaccination with ΔSHrBRSV failed to induce a detectable immunological response, there was a rapid and strong anamnestic mucosal BRSV-specific IgA, virus neutralizing antibody and local T cell response following challenge with virulent BRSV. Calves immunized twice intramuscularly, three weeks apart with SUMont were also well protected two weeks after boost. The protection was not as pronounced as that in ΔSHrBRSV-immunized animals, but superior to those immunized twice subcutaneously three weeks apart with SUAbis. Antibody responses induced by the subunit vaccines were non-neutralizing and not directed against BRSV F or G proteins. When formulated as SUMont but not as SUAbis, the HRSV N, P and M2-1 proteins induced strong systemic cross-protective cell-mediated immune responses detectable already after priming. ΔSHrBRSV and SUMont are two promising DIVA-compatible vaccines, apparently inducing protection by

  10. Different forms of MARCKS protein are involved in memory formation in the learning process of imprinting.

    Science.gov (United States)

    Solomonia, Revaz O; Apkhazava, David; Nozadze, Maia; Jackson, Antony P; McCabe, Brian J; Horn, Gabriel

    2008-06-01

    There is strong evidence that a restricted part of the chick forebrain, the IMM (formerly IMHV), stores information acquired through the learning process of visual imprinting. Twenty-four hours after imprinting training, a learning-specific increase in amount of myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is known to occur in the homogenate fraction of IMM. We investigated the two components of this fraction, membrane-bound and cytoplasmic-phosphorylated MARCKS. In IMM, amount of membrane-bound MARCKS, but not of cytoplasmic-phosphorylated MARCKS, increased as chicks learned. No changes were observed for either form of MARCKS in PPN, a control forebrain region. The results indicate that there is a learning-specific increase in membrane-bound, non-phosphorylated MARCKS 24 h after training. This increase might contribute to stabilization of synaptic morphology.

  11. HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity.

    Science.gov (United States)

    Hategan, Alina; Bianchet, Mario A; Steiner, Joseph; Karnaukhova, Elena; Masliah, Eliezer; Fields, Adam; Lee, Myoung-Hwa; Dickens, Alex M; Haughey, Norman; Dimitriadis, Emilios K; Nath, Avindra

    2017-02-20

    Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

  12. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

  13. The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation.

    Science.gov (United States)

    Rogala, Kacper B; Dynes, Nicola J; Hatzopoulos, Georgios N; Yan, Jun; Pong, Sheng Kai; Robinson, Carol V; Deane, Charlotte M; Gönczy, Pierre; Vakonakis, Ioannis

    2015-05-29

    Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

  14. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

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    Luthey-Schulten Zaida

    2009-07-01

    Full Text Available Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding and C- (zinc-free variants, with 26 genomes (mainly from the class Clostridia containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution

  15. Effect of organophosphorus insecticides on phosphorylation of the M2 muscarinic acetylcholine receptor

    Institute of Scientific and Technical Information of China (English)

    Shuyin Li; Liming Zou; Carry Pope

    2008-01-01

    BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2.OBJECTIVE: To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides.DESIGN, TIME AND SETTING: An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004.METHODS: The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [OP32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter.MAIN OUTCOME MEASURES: Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the adrenergic receptor.CONCLUSION: Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein

  16. Kinetic study of coniferyl alcohol radical binding to the (+)-pinoresinol forming dirigent protein.

    Science.gov (United States)

    Halls, Steven C; Davin, Laurence B; Kramer, David M; Lewis, Norman G

    2004-03-01

    An essential step in lignan and lignin formation in planta is one electron oxidation of (E)-coniferyl alcohol (CA) to generate the radical intermediate (CA(*)), which can then undergo directed radical-radical couplings in vivo. For lignan formation in vitro and in vivo, stereoselective coupling of CA(*) only occurs to afford (+)-pinoresinol in the additional presence of (+)-pinoresinol forming dirigent protein (DP). Presented herein is a kinetic and thermodynamic study which reveals the central mechanistic details of the coupling process involved in DP-mediated coupling. DP activity was maximal between pH 4.25 and pH 6.0, with activity being maintained at temperatures below 33 degrees C. Equilibrium binding assays revealed that coniferyl alcohol was only weakly bound to the DP, with a K(D) of 370 +/- 65 microM. On the other hand, the enantiomeric excess of (+)-pinoresinol formed was dependent on both DP concentration and rate of CA oxidation and, thus, on apparent steady-state [CA(*)]. The data obtained could best be explained using a kinetic model where radical-radical coupling via DP competes with that occurring in open solution. Using this model, an apparent K(M) of about 10 nM was estimated from the saturation behavior of (+)-pinoresinol formation with respect to apparent steady-state [CA(*)]. These data strongly suggest that CA(*), rather than CA, is the substrate for DP, in agreement with earlier predictions. A mechanism of directed radical-radical coupling, where two coniferyl alcohol radical substrates are bound per protein dimer, is proposed.

  17. Bacterial collagen-like proteins that form triple-helical structures.

    Science.gov (United States)

    Yu, Zhuoxin; An, Bo; Ramshaw, John A M; Brodsky, Barbara

    2014-06-01

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

  18. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.

    Science.gov (United States)

    De Colibus, Luigi; Sonnen, Andreas F-P; Morris, Keith J; Siebert, C Alistair; Abrusci, Patrizia; Plitzko, Jürgen; Hodnik, Vesna; Leippe, Matthias; Volpi, Emanuela; Anderluh, Gregor; Gilbert, Robert J C

    2012-09-05

    Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bar-Kana, I.; Pitaru, S. [Tel Aviv Univ., Dept. of Oral Biology, Goldschleger School of dental Medicine (Israel); Savion, N. [Tel Aviv Univ., Goldschleger Eye Research Inst. (Israel); Narayanan, A.S. [Univ. of Washington, Dept. of Pathology, Faculty of Medicine (United States)

    1998-08-01

    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 {sup 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) 39 refs.

  20. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.

    Science.gov (United States)

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-10-16

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

  1. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

    Science.gov (United States)

    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

    2015-07-01

    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  2. 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...LVGRHSRKPWLKFMNADNQHLVSPEAIDFLDKLLRYDHQERLTALEAMTHPYFQQVRAAENSRTRA corn_1M2Q.jpg ...

  3. Main: 1M2P [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1M2P トウモロコシ Corn Zea mays L. Casein Kinase Ii, Alpha Chain Name=Ack2; Zea Mays Mole...LVGRHSRKPWLKFMNADNQHLVSPEAIDFLDKLLRYDHQERLTALEAMTHPYFQQVRAAENSRTRA corn_1M2P.jpg ...

  4. Main: 1M2R [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1M2R トウモロコシ Corn Zea mays L. Casein Kinase Ii, Alpha Chain Name=Ack2; Zea Mays Mole...ELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNHDQLVKIAKVLGTDGLNVYLNKYRIELDPQLEALVGRHSRKPWLKFMNADNQHLVSPEAIDFLDKLLRYDHQERLTALEAMTHPYFQQVRAAENSRTRA corn_1M2R.jpg ...

  5. 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.

  6. Effects of bimetallic doping on small cyclic and tubular boron clusters: B7M2 and B14M2 structures with M = Fe, Co.

    Science.gov (United States)

    Pham, Hung Tan; Nguyen, Minh Tho

    2015-07-14

    Using density functional theory with the TPSSh functional and the 6-311+G(d) basis set, we extensively searched for the global minima of two metallic atoms doped boron clusters B6M2, B7M2, B12M2 and B14M2 with transition metal element M being Co and Fe. Structural identifications reveal that B7Co2, B7Fe2 and B7CoFe clusters have global minima in a B-cyclic motif, in which a perfectly planar B7 is coordinated with two metallic atoms placed along the C7 axis. The B6 cluster is too small to form a cycle with the presence of two metals. Similarly, the B12 cluster is not large enough to stabilize the metallic dimer within a double ring 2 × B6 tube. The doped B14M2 clusters including B14Co2, B14Fe2 and B14CoFe have a double ring 2 × B7 tubular shape in which one metal atom is encapsulated by the B14 tube and the other is located at an exposed position. Dissociation energies demonstrate that while bimetallic cyclic cluster B7M2 prefers a fragmentation channel that generates the B7 global minimum plus metallic dimer, the tubular structure B14M2 tends to dissociate giving a bimetallic cyclic structure B7M2 and a B@B6 cluster. The enhanced stability of the bimetallic doped boron clusters considered can be understood from the stabilizing interactions between the anti-bonding MOs of metal-metal dimers and the levels of a disk aromatic configuration (for bimetallic cyclic structures), or the eigenstates of the B14 tubular form (in case of bimetallic tubular structure).

  7. Structural Features of Apicomplexan Pore-Forming Proteins and Their Roles in Parasite Cell Traversal and Egress.

    Science.gov (United States)

    Guerra, Alfredo J; Carruthers, Vern B

    2017-08-29

    Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. This review highlights recent insight gained from the newly available three-dimensional structures of several known or putative apicomplexan pore-forming proteins that contribute to cell traversal or egress. These new structural advances suggest that parasite pore-forming proteins use distinct mechanisms to disrupt host cell membranes at multiple steps in parasite life cycles. How proteolytic processing, secretion, environment, and the accessibility of lipid receptors regulate the membranolytic activities of such proteins is also discussed.

  8. The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

    Science.gov (United States)

    Figueroa, Maximiliano; Sleutel, Mike; Vandevenne, Marylene; Parvizi, Gregory; Attout, Sophie; Jacquin, Olivier; Vandenameele, Julie; Fischer, Axel W; Damblon, Christian; Goormaghtigh, Erik; Valerio-Lepiniec, Marie; Urvoas, Agathe; Durand, Dominique; Pardon, Els; Steyaert, Jan; Minard, Philippe; Maes, Dominique; Meiler, Jens; Matagne, André; Martial, Joseph A; Van de Weerdt, Cécile

    2016-07-01

    Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

  9. DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

    Science.gov (United States)

    Carra, Claudio; Cucinotta, Francis A.

    2007-01-01

    Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

  10. 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.

  11. A nacre protein, n16.3, self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.

    Science.gov (United States)

    Perovic, Iva; Chang, Eric P; Lui, Michael; Rao, Ashit; Cölfen, Helmut; Evans, John Spencer

    2014-04-29

    The mollusk shell is a complex biological material that integrates mineral phases with organic macromolecular components such as proteins. The role of proteins in the formation of the nacre layer (aragonite mineral phase) is poorly understood, particularly with regard to the organization of mineral deposits within the protein extracellular matrix and the identification of which proteins are responsible for this task. We report new experiments that provide insight into the role of the framework nacre protein, n16.3 (Pinctada fucata), as an organizer or assembler of calcium carbonate mineral clusters. Using a combination of biophysical techniques, we find that recombinant n16.3 (r-n16.3) oligomerizes to form amorphous protein films and particles that possess regions of disorder and mobility. These supramolecular assemblies possess an intrinsically disordered C-terminal region (T64-W98) and reorganize in the presence of Ca(2+) ions to form clustered protein oligomers. This Ca(2+)-induced reorganization leads to alterations in the molecular environments of Trp residues, the majority of which reside in putative aggregation-prone cross-β strand regions. Potentiometric Ca(2+) titrations reveal that r-n16.3 does not significantly affect the formation of prenucleation clusters in solution, and this suggests a role for this protein in postnucleation mineralization events. This is verified in subsequent in vitro mineralization assays in which r-n16.3 demonstrates its ability to form gel-like protein phases that organize and cluster nanometer-sized single-crystal calcite relative to protein-deficient controls. We conclude that the n16 nacre framework proteome creates a protein gel matrix that organizes and dimensionally limits mineral deposits. This process is highly relevant to the formation of ordered, nanometer-sized nacre tablets in the mollusk shell.

  12. 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.

  13. The HhH domain of the human DNA repair protein XPF forms stable homodimers.

    Science.gov (United States)

    Das, Devashish; Tripsianes, Konstantinos; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2008-03-01

    The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.

  14. Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy.

    Science.gov (United States)

    Mandala, Venkata S; Liao, Shu-Yu; Kwon, Byungsu; Hong, Mei

    2017-07-07

    The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus. (15)N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Microscopic relaxations in a protein sustained down to 160K in a non-glass forming organic solvent.

    Science.gov (United States)

    Mamontov, E; O'Neill, H

    2017-01-01

    We have studied microscopic dynamics of a protein in carbon disulfide, a non-glass forming solvent, down to its freezing temperature of ca. 160K. We have utilized quasielastic neutron scattering. A comparison of lysozyme hydrated with water and dissolved in carbon disulfide reveals a stark difference in the temperature dependence of the protein's microscopic relaxation dynamics induced by the solvent. In the case of hydration water, the common protein glass-forming solvent, the protein relaxation slows down in response to a large increase in the water viscosity on cooling down, exhibiting a well-known protein dynamical transition. The dynamical transition disappears in non-glass forming carbon disulfide, whose viscosity remains a weak function of temperature all the way down to freezing at just below 160K. The microscopic relaxation dynamics of lysozyme dissolved in carbon disulfide is sustained down to the freezing temperature of its solvent at a rate similar to that measured at ambient temperature. Our results demonstrate that protein dynamical transition is not merely solvent-assisted, but rather solvent-induced, or, more precisely, is a reflection of the temperature dependence of the solvent's glass-forming dynamics. We hypothesize that, if the long debated idea regarding the direct link between the microscopic relaxations and the biological activity in proteins is correct, then not only the microscopic relaxations, but also the activity, could be sustained in proteins all the way down to the freezing temperature of a non-glass forming solvent with a weak temperature dependence of its viscosity. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Designing inhibitors of M2 proton channel against H1N1 swine influenza virus.

    Directory of Open Access Journals (Sweden)

    Qi-Shi Du

    Full Text Available BACKGROUND: M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591-595 may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus. METHODOLOGY: Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors. CONCLUSIONS: 1 The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2 The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3 The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a "barrel hoop", holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4 The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.

  17. 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.

  18. The fungal cerato-platanin protein EPL1 forms highly ordered layers at hydrophobic/hydrophilic interfaces.

    Science.gov (United States)

    Bonazza, K; Gaderer, R; Neudl, S; Przylucka, A; Allmaier, G; Druzhinina, I S; Grothe, H; Friedbacher, G; Seidl-Seiboth, V

    2015-03-07

    Cerato-platanin proteins (CPPs) and hydrophobins are two classes of small, secreted proteins that are exclusively found in fungi. CPPs are known as chitin-binding proteins, and were recently also shown to form protein layers at air/water interfaces, but the features of these layers were not investigated on the molecular level yet. In this study, by means of atomic force microscopy (AFM), EPL1, a member of the CPP family was shown to form highly ordered monolayers at a hydrophobic surface/liquid-interface. Furthermore, two new hydrophobins were analysed, and the influence of EPL1 on hydrophobin layers was studied in situ. Hydrophobins are amphiphilic proteins that are able to self-assemble at hydrophobic/hydrophilic interfaces, thereby inverting the polarity of the surface. This renders fungal growth structures such as spores water repellent. The combination of AFM data and wettability experiments led to the conclusion that in presence of both, hydrophobins and EPL1, a previously unknown hybrid layer is formed. This mixed protein layer is on one hand not inverting but enhancing the hydrophobicity of HOPG (highly oriented pyrolytic graphite), typical for EPL1, and on the other hand, it is stable and water insoluble, which is reminiscent of hydrophobin layers.

  19. The protein folds as platonic forms: new support for the pre-Darwinian conception of evolution by natural law.

    Science.gov (United States)

    Denton, Michael J; Marshall, Craig J; Legge, Michael

    2002-12-07

    Before the Darwinian revolution many biologists considered organic forms to be determined by natural law like atoms or crystals and therefore necessary, intrinsic and immutable features of the world order, which will occur throughout the cosmos wherever there is life. The search for the natural determinants of organic form-the celebrated "Laws of Form"-was seen as one of the major tasks of biology. After Darwin, this Platonic conception of form was abandoned and natural selection, not natural law, was increasingly seen to be the main, if not the exclusive, determinant of organic form. However, in the case of one class of very important organic forms-the basic protein folds-advances in protein chemistry since the early 1970s have revealed that they represent a finite set of natural forms, determined by a number of generative constructional rules, like those which govern the formation of atoms or crystals, in which functional adaptations are clearly secondary modifications of primary "givens of physics." The folds are evidently determined by natural law, not natural selection, and are "lawful forms" in the Platonic and pre-Darwinian sense of the word, which are bound to occur everywhere in the universe where the same 20 amino acids are used for their construction. We argue that this is a major discovery which has many important implications regarding the origin of proteins, the origin of life and the fundamental nature of organic form. We speculate that it is unlikely that the folds will prove to be the only case in nature where a set of complex organic forms is determined by natural law, and suggest that natural law may have played a far greater role in the origin and evolution of life than is currently assumed.

  20. Kinked structures of isolated nicotinic receptor M2 helices: a molecular dynamics study.

    Science.gov (United States)

    Sankararamakrishnan, R; Samsom, M S

    1994-12-01

    The pore-lining M2 helix of the nicotinic acetylcholine receptor exhibits a pronounced kink when the corresponding ion channel is in a closed conformation [N. Unwin (1993) Journal of Molecular Biology, Vol. 229, pp. 1101-1124]. We have performed molecular dynamics simulations of isolated 22-residue M2 helices in order to identify a possible molecular origin of this kink. In order to sample a wide range of conformational space, a simulated annealing protocol was used to generate five initial M2 helix structures, each of which was subsequently used as the basis of 300 ps MD simulations. Two helix sequences (M2 alpha and M2 delta) were studied in this manner, resulting in a total of ten 300 ps trajectories. Kinked helices present in the trajectories were identified and energy minimized to yield a total of five different stable kinked structures. For comparison, a similar molecular dynamics simulation of a Leu23 helix yielded no stable kinked structures. In four of the five kinked helices, the kink was stabilized by H bonds between the helix backbone and polar side-chain atoms. Comparison with data from the literature on site-directed mutagenesis of M2 residues suggests that such polar side-chain to main-chain H bonds may also contribute to kinking of M2 helices in the intact channel protein.

  1. 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.

  2. The Demand for Divisia M2 in China%中国Divisa M2需求模型

    Institute of Scientific and Technical Information of China (English)

    潘红宇; 邓述慧

    2001-01-01

    本文计算中国Divisa M2指数并对其建立需求模型,研究发现实际Divisa M2 与实际产出存在协整关系,采用误差校正方法建立的动态需求模型具有良好的稳定性。%This paper computes the Divisa M2 in China and makes the money demand model for it. The research finds that the demand for real Divisa M2 is cointegrated with the real output, the short-run model is stable also.

  3. 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.

  4. 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

  5. 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

    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...

  6. 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 enzymati

  7. 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 enzymati

  8. 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 ...

  9. 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.

  10. Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets.

    Science.gov (United States)

    Han, Linjie; Ruotolo, Brandon T

    2015-07-01

    Electrospray ionization coupled to mass spectrometry is a key technology for determining the stoichiometries of multiprotein complexes. Despite highly accurate results for many assemblies, challenging samples can generate signals for artifact protein-protein binding born of the crowding forces present within drying electrospray droplets. Here, for the first time, we study the formation of preferred protein quaternary structures within such rapidly evaporating nanodroplets. We use ion mobility and tandem mass spectrometry to investigate glutamate dehydrogenase dodecamers and serum amyloid P decamers as a function of protein concentration, along with control experiments using carefully chosen protein analogues, to both establish the formation of operative mechanisms and assign the bimodal conformer populations observed. Further, we identify an unprecedented symmetric collision-induced dissociation pathway that we link directly to the quaternary structures of the precursor ions selected.

  11. Water-mediated conformational transitions in nicotinic receptor M2 helix bundles: a molecular dynamics study.

    Science.gov (United States)

    Sankararamakrishnan, R; Sansom, M S

    1995-12-27

    The ion channel of the nicotinic acetylcholine receptor is a water-filled pore formed by five M2 helix segments, one from each subunit. Molecular dynamics simulations on bundles of five M2 alpha 7 helices surrounding a central column of water and with caps of water molecules at either end of the pore have been used to explore the effects of intrapore water on helix packing. Interactions of water molecules with the N-terminal polar sidechains lead to a conformational transition from right- to left-handed supercoils during these stimulations. These studies reveal that the pore formed by the bundle of M2 helices is flexible. A structural role is proposed for water molecules in determining the geometry of bundles of isolated pore-forming helices.

  12. 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)

  13. 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

  14. A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

    Science.gov (United States)

    Barchad-Avitzur, Ofra; Priest, Michael F; Dekel, Noa; Bezanilla, Francisco; Parnas, Hanna; Ben-Chaim, Yair

    2016-10-04

    G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

  15. Trypsin, Tryptase, and Thrombin Polarize Macrophages towards a Pro-Fibrotic M2a Phenotype.

    Directory of Open Access Journals (Sweden)

    Michael J V White

    Full Text Available For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages.

  16. Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

    Directory of Open Access Journals (Sweden)

    Phuong Nguyen

    2016-01-01

    Full Text Available We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor by DTor-specific inhibitory double-stranded RNA (RNAi induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH. Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved.

  17. Discovery of Potential M2 Channel Inhibitors Based on the Amantadine Scaffold via Virtual Screening and Pharmacophore Modeling

    Directory of Open Access Journals (Sweden)

    Ly Le

    2011-12-01

    Full Text Available The M2 channel protein on the influenza A virus membrane has become the main target of the anti-flu drugs amantadine and rimantadine. The structure of the M2 channel proteins of the H3N2 (PDB code 2RLF and 2009-H1N1 (Genbank accession number GQ385383 viruses may help researchers to solve the drug-resistant problem of these two adamantane-based drugs and develop more powerful new drugs against influenza A virus. In the present study, we searched for new M2 channel inhibitors through a combination of different computational methodologies, including virtual screening with docking and pharmacophore modeling. Virtual screening was performed to calculate the free energies of binding between receptor M2 channel proteins and 200 new designed ligands. After that, pharmacophore analysis was used to identify the important M2 protein-inhibitor interactions and common features of top binding compounds with M2 channel proteins. Finally, the two most potential compounds were determined as novel leads to inhibit M2 channel proteins in both H3N2 and 2009-H1N1 influenza A virus.

  18. Discovery of potential M2 channel inhibitors based on the amantadine scaffold via virtual screening and pharmacophore modeling.

    Science.gov (United States)

    Tran, Linh; Choi, Sy Bing; Al-Najjar, Belal O; Yusuf, Muhammad; Wahab, Habibah A; Le, Ly

    2011-12-08

    The M2 channel protein on the influenza A virus membrane has become the main target of the anti-flu drugs amantadine and rimantadine. The structure of the M2 channel proteins of the H3N2 (PDB code 2RLF) and 2009-H1N1 (Genbank accession number GQ385383) viruses may help researchers to solve the drug-resistant problem of these two adamantane-based drugs and develop more powerful new drugs against influenza A virus. In the present study, we searched for new M2 channel inhibitors through a combination of different computational methodologies, including virtual screening with docking and pharmacophore modeling. Virtual screening was performed to calculate the free energies of binding between receptor M2 channel proteins and 200 new designed ligands. After that, pharmacophore analysis was used to identify the important M2 protein-inhibitor interactions and common features of top binding compounds with M2 channel proteins. Finally, the two most potential compounds were determined as novel leads to inhibit M2 channel proteins in both H3N2 and 2009-H1N1 influenza A virus.

  19. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    Science.gov (United States)

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  20. Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

    Institute of Scientific and Technical Information of China (English)

    Chao FAN; Lan-lan ZHANG; Cheng-feng LEI; Qin FANG

    2009-01-01

    Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with uNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the uNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335.742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335.742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

  1. 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.

  2. Carboxyl- and amino-functionalized polystyrene nanoparticles differentially affect the polarization profile of M1 and M2 macrophage subsets.

    Science.gov (United States)

    Fuchs, Ann-Kathrin; Syrovets, Tatiana; Haas, Karina A; Loos, Cornelia; Musyanovych, Anna; Mailänder, Volker; Landfester, Katharina; Simmet, Thomas

    2016-04-01

    Macrophages are key regulators of innate and adaptive immune responses. Exposure to microenvironmental stimuli determines their polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. M1 exhibit high expression of proinflammatory TNF-α and IL-1β, and M2 promote tissue repair, but likewise support tumor growth and cause immune suppression by expressing IL-10. Thus, the M1/M2 balance critically determines tissue homeostasis. By using carboxyl- (PS-COOH) and amino-functionalized (PS-NH2) polystyrene nanoparticles, the effects of surface decoration on the polarization of human macrophages were investigated. The nanoparticles did not compromise macrophage viability nor did they affect the expression of the M1 markers CD86, NOS2, TNF-α, and IL-1β. By contrast, in M2, both nanoparticles impaired expression of scavenger receptor CD163 and CD200R, and the release of IL-10. PS-NH2 also inhibited phagocytosis of Escherichia coli by both, M1 and M2. PS-COOH did not impair phagocytosis by M2, but increased protein mass in M1 and M2, TGF-β1 release by M1, and ATP levels in M2. Thus, nanoparticles skew the M2 macrophage polarization without affecting M1 markers. Given the critical role of the M1 and M2 polarization for the immunological balance in patients with cancer or chronic inflammation, functionalized nanoparticles might serve as tools for reprogramming the M1/M2 polarization.

  3. 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.

  4. Multiple Access Technologies for Cellular M 2M Communications

    Institute of Scientific and Technical Information of China (English)

    Mahyar Shirvanimoghaddam; Sarah J. Johnson

    2016-01-01

    This paper reviews the multiple access techniques for machine⁃to⁃machine (M2M) communications in future wireless cellular net⁃works. M2M communications aims at providing the communication infrastructure for the emerging Internet of Things (IoT), which will revolutionize the way we interact with our surrounding physical environment. We provide an overview of the multiple access strategies and explain their limitations when used for M2M communications. We show the throughput efficiency of different multi⁃ple access techniques when used in coordinated and uncoordinated scenarios. Non⁃orthogonal multiple access (NOMA) is also shown to support a larger number of devices compared to orthogonal multiple access techniques, especially in uncoordinated sce⁃narios. We also detail the issues and challenges of different multiple access techniques to be used for M2M applications in cellu⁃lar networks.

  5. 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.

  6. Muscarinic cholinergic receptor (M2 plays a crucial role in the development of myopia in mice

    Directory of Open Access Journals (Sweden)

    Veluchamy A. Barathi

    2013-09-01

    Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2 were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error.

  7. M2-Edge Colorings Of Cacti And Graph Joins

    Directory of Open Access Journals (Sweden)

    Czap Július

    2016-02-01

    Full Text Available An edge coloring φ of a graph G is called an M2-edge coloring if |φ(v| ≤ 2 for every vertex v of G, where φ(v is the set of colors of edges incident with v. Let 2(G denote the maximum number of colors used in an M2-edge coloring of G. In this paper we determine 2(G for trees, cacti, complete multipartite graphs and graph joins.

  8. Heat-induced destabilization of oil-in-water emulsions formed from hydrolyzed whey protein.

    Science.gov (United States)

    Euston, S R; Finnigan, S R; Hirst, R L

    2001-11-01

    The emulsifying ability, heat stability, and coalescence stability of oil-in-water emulsions prepared with whey protein of varied degrees of hydrolysis (DH), and at varied protein contents, was studied. Whey protein hydrolysates (WPH) with a DH of 4% and 10% had poorer emulsifying ability than non-hydrolyzed whey protein concentrate (WPC), but were more heat stable. Increasing DH between 10 and 27% improved emulsifying ability and further improved the heat stability of the emulsion droplets. Increasing DH from 27 to 35% led to a big decrease in both emulsifying ability and heat stability. The quiescent coalescence stability of WPH emulsions was relatively good up to a DH of 27%. Above DH 27% emulsions become highly unstable. It appears that two mechanisms of instability are at work here. At low DH heat-induced denaturation and aggregation occur. In the DH range of 4-20% heat stability increases as protein globular structure is disrupted. At a DH greater than 27% we see a change from a hydrolysis-induced increase in heat-stability to coalescence instability, with a resultant large increase in emulsion breakdown during heating.

  9. 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.

  10. Study of {sup 179}Hf{sup m2} excitation

    Energy Technology Data Exchange (ETDEWEB)

    Vishnevsky, I. N.; Zheltonozhsky, V. A., E-mail: zhelton@kinr.kiev.ua; Savrasov, A. N. [National Academy of Sciences of Ukraine, Institute for Nuclear Research (Ukraine); Mazur, V. M. [National Academy of Sciences of Ukraine, Institute of Electronic Physics (Ukraine)

    2016-12-15

    Isomeric ratios of {sup 179}Hf{sup m2,g} yields in the (γ, n) reaction and the cross section for the {sup 179}Hf{sup m2} population in the (α, p) reaction are measured for the first time at the end-point energies of 15.1 and 17.5 MeV for bremsstrahlung photons and 26 MeV for alpha particles. The results are σ = (1.1 ± 0.11) × 10{sup −27} cm{sup 2} for the {sup 176}Lu(α, p){sup 179}Hf{sup m2} reaction and Y{sub m2}/Y{sub g} = (6.1 ± 0.3) × 10{sup −6} and (3.7 ± 0.2) × 10{sup −6} for the {sup 180}Hf(γ, n){sup 179}Hf{sup m22} reaction at E{sub ep} =15.1 and 17.5 MeV, respectively. The experimental data on the relative {sup 179}Hf{sup m2} yield indicate a single-humped shape of the excitation function for the {sup 180}Hf(γ, n){sup 179}Hf{sup m2} reaction. Simulation is performed using the TALYS-1.4 and EMPIRE-3.2 codes.

  11. PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin.

    Science.gov (United States)

    Sakono, Masafumi; Seko, Akira; Takeda, Yoichi; Ito, Yukishige

    2014-09-12

    Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57-calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.

  12. Comparison of the effect of homocysteine in the reduced form, its thiolactone and protein homocysteinylation on hemostatic properties of plasma.

    Science.gov (United States)

    Malinowska, Joanna; Nowak, Paweł; Olas, Beata

    2011-03-01

    Mechanisms involved in the relationship between hyperhomocysteinemia and hemostatic process are still unclear. In the literature there are few papers describing studies on the effects of homocysteine (Hcys) on proteins that participate in blood coagulation and fibrinolysis in human. The aim of our study was to establish and compare the influence of a reduced form of Hcys (at final doses of 0.01 - 1 mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 0.1 - 1 μM) on the clot formation (using whole human plasma and purified fibrinogen) and the fibrin lysis. Moreover, the aim of our study was to explain the effect of plasma protein modifications (S- and N-homocysteinylation) on selected parameters of hemostasis. We observed that HTL, like its precursor, a reduced form of Hcys stimulated polymerization of fibrinogen, but this process was not dose-dependent. In the presence of HTL (at the lowest tested concentration - 0.1μM) the increase was about 55%. Our present results also demonstrated that Hcys in the reduced form (0.01 - 1 mM) and HTL at lower doses than Hcys (0.1 - 1 μM) reduced the fibrin lysis in whole human plasma. Our results reported that HTL, like the reduced form of Hcys (at concentrations corresponding to concentrations in plasma during hyperhomocysteinemia) induced modifications of hemostatic plasma proteins, and the consequence of these modifications may be alteration in protein structure associated with changes of hemostatic functions.

  13. 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.

  14. 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 various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP...

  15. Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2007-02-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV, a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. Results The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. Conclusion The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.

  16. Conformational response of influenza A M2 transmembrane domain to amantadine drug binding at low pH (pH 5.5

    Directory of Open Access Journals (Sweden)

    Elka R. Georgieva

    2016-07-01

    Full Text Available The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21-49 fragment, upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR spectroscopy and electron microscopy were applied to M2TMD21-49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21-49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21-49. As reported by double electron-electron resonance (DEER, in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L’s ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels by 5-8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD21-49 through increased motional ordering. In contrast to wild-type M2TMD21-49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation

  17. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    Science.gov (United States)

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  18. Solution structure of the pore-forming protein of Entamoeba histolytica

    NARCIS (Netherlands)

    Hecht, O.; van Nuland, N.A.J.; Schleinkofer, K.; Dingley, A.J.; Bruhn, H.; Leippe, M.; Grötzinger, J.

    2004-01-01

    Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica. Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic di

  19. Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba

    NARCIS (Netherlands)

    Michalek, M.; Sönnichsen, F.D.; Wechselberger, R.W.; Wienk, H.L.J.; Leippe, M.; et al., [No Value

    2013-01-01

    Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acantha

  20. Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba

    NARCIS (Netherlands)

    Michalek, M.; Sönnichsen, F.D.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; Wienk, H.L.J.|info:eu-repo/dai/nl/203884884; Leippe, M.; et al., [No Value

    2013-01-01

    Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize

  1. In situ forming biodegradable hydrogels and their application for protein delivery

    NARCIS (Netherlands)

    Hiemstra, Christine

    2007-01-01

    Hydrogels have been widely applied for biomedical applications, such as protein delivery and tissue engineering, due to their similarity with the extracellular matrix. Hydrogels are water-swollen, insoluble polymer networks. Their high water content renders them compatible with living tissue and pro

  2. 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

    expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane...

  3. 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.

  4. 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.

  5. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P;

    1994-01-01

    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  6. 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

  7. M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation.

    Science.gov (United States)

    Morgan, Hugh P; O'Reilly, Francis J; Wear, Martin A; O'Neill, J Robert; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

    2013-04-09

    We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC50 value of 0.24 mM, whereas thyroid hormone (triiodo-L-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC50 (concentration that gives 50% activation) of 7 μM] shifts the equilibrium to the tetrameric active R-state, which has a similar activity to that of the constitutively fully active isoform M1PYK. Proliferation assays using HCT-116 cells showed that addition of inhibitors phenylalanine and T3 both increased cell proliferation, whereas addition of the activator F16BP reduced proliferation. F16BP abrogates the inhibitory effect of both phenylalanine and T3, highlighting a dominant role of M2PYK allosteric activation in the regulation of cancer proliferation. X-ray structures show constitutively fully active M1PYK and F16BP-bound M2PYK in an R-state conformation with a lysine at the dimer-interface acting as a peg in a hole, locking the active tetramer conformation. Binding of phenylalanine in an allosteric pocket induces a 13° rotation of the protomers, destroying the peg-in-hole R-state interface. This distinct T-state tetramer is stabilized by flipped out Trp/Arg side chains that stack across the dimer interface. X-ray structures and biophysical binding data of M2PYK complexes explain how, at a molecular level, fluctuations in concentrations of amino acids, thyroid hormone, and glucose metabolites switch M2PYK on and off to provide the cell with a nutrient sensing and growth signaling mechanism.

  8. 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

    remission rate (73% vs 75%; odds ratio, 1.07 [0.83-1.39]; P = .6) or in any recognized subgroup. The 60-day mortality was increased in the 90 mg/m(2) arm (10% vs 5% (hazard ratio [HR] 1.98 [1.30-3.02]; P = .001), which resulted in no difference in overall 2-year survival (59% vs 60%; HR, 1.16 [0.95-1.43]; P...... recommended as a standard of care. However, 60 mg/m(2) is widely used and has never been directly compared with 90 mg/m(2). As part of the UK National Cancer Research Institute (NCRI) AML17 trial, 1206 adults with untreated AML or high-risk myelodysplastic syndrome, mostly younger than 60 years of age, were...... randomized to a first-induction course of chemotherapy, which delivered either 90 mg/m(2) or 60 mg/m(2) on days 1, 3, and 5 combined with cytosine arabinoside. All patients then received a second course that included daunorubicin 50 mg/m(2) on days 1, 3, and 5. There was no overall difference in complete...

  9. 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.

  10. Modelling packing interactions in parallel helix bundles: pentameric bundles of nicotinic receptor M2 helices.

    Science.gov (United States)

    Sankararamakrishnan, R; Sansom, M S

    1995-11-01

    The transbilayer pore of the nicotinic acetylcholine receptor (nAChR) is formed by a pentameric bundle of M2 helices. Models of pentameric bundles of M2 helices have been generated using simulated annealing via restrained molecular dynamics. The influence of: (a) the initial C alpha template; and (b) screening of sidechain electrostatic interactions on the geometry of the resultant M2 helix bundles is explored. Parallel M2 helices, in the absence of sidechain electrostatic interactions, pack in accordance with simple ridges-in-grooves considerations. This results in a helix crossing angle of ca. +12 degrees, corresponding to a left-handed coiled coil structure for the bundle as a whole. Tilting of M2 helices away from the central pore axis at their C-termini and/or inclusion of sidechain electrostatic interactions may perturb such ridges-in-grooves packing. In the most extreme cases right-handed coiled coils are formed. An interplay between inter-helix H-bonding and helix bundle geometry is revealed. The effects of changes in electrostatic screening on the dimensions of the pore mouth are described and the significance of these changes in the context of models for the nAChR pore domain is discussed.

  11. [Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

    Science.gov (United States)

    Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

    2012-01-01

    Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

  12. [Comparative study of the protein makeup in diploid and haploid forms of Saccharomyces and Pichia].

    Science.gov (United States)

    Temina, A V; Tolstorukov, I I; Korogodin, V I; Gololobov, A D

    1979-01-01

    The rates of growth, biomass accumulation, and electrophoretic spectra of mobile cytoplasmic proteins were studied with nonisogenous haploid and diploid cultures of Saccharomyces cerevisiae and Pichia guilliermondii as well as with isogenous haploid-diploid pairs of Saccharomyces cerevisiae and Pichia pinus. On a mineral medium with glucose, differences in these parameters in various yeast strains were found to be due to the genotype of a strain rather than to ploidy: nonisogenous haploid and diploid cultures displayed considerable and random variability of these properties while no differences were found in isogenous haploid-diploid pairs. Studies on solubility of protein fractions in various solvents made it possible to reveal differences connected with ploidy, namely: both in nonisogenous and isogenous haploid-diploid systems, the content of the water-soluble fraction decreased in diploid cultures.

  13. Structural characterization of yeast acidic ribosomal P proteins forming the P1A-P2B heterocomplex.

    Science.gov (United States)

    Tchórzewski, Marek; Krokowski, Dawid; Boguszewska, Aleksandra; Liljas, Anders; Grankowski, Nikodem

    2003-04-01

    Acidic ribosomal P proteins form a distinct lateral protuberance on the 60S ribosomal subunit. In yeast, this structure is composed of two heterocomplexes (P1A-P2B and P1B-P2A) attached to the ribosome with the aid of the P0 protein. In solution, the isolated P proteins P1A and P2B have a flexible structure with some characteristics of a molten globule [Zurdo, J., et al. (2000) Biochemistry 39, 8935-8943]. In this report, the structure of P1A-P2B heterocomplex from Saccharomyces cerevisiae is investigated by means of size-exclusion chromatography, chemical cross-linking, circular dichroism, light scattering, and fluorescence spectroscopy. The circular dichroism experiment shows that the complex could be ranked in the tertiary class of all-alpha proteins, with an average alpha-helical content of approximately 65%. Heat and urea denaturation experiments reveal that the P1A-P2B complex, unlike the isolated proteins, has a full cooperative transition which can be fitted into a two-state folding-unfolding model. The behavior of the complex in the presence of 2,2,2-trifluoroethanol also resembles a two-state folding-unfolding transition, further supporting the idea that the heterocomplex contains well-packed side chains. In conclusion, the P1A-P2B heterocomplex, unlike the isolated proteins, has a well-defined hydrophobic core. Consequently, the complex can put up its structure without additional ribosomal components, so the heterodimeric complex reflects the intrinsic properties of the two analyzed proteins, indicating thus that this is the only possible configuration of the P1A and P2B proteins on the ribosomal stalk structure.

  14. 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.

  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

    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.

  17. 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.

  18. M$^2$I Communication: From Theoretical Modeling to Practical Design

    CERN Document Server

    Guo, Hongzhi

    2015-01-01

    Wireless communications in complex environments are constrained by lossy media and complicated structures. Magnetic Induction (MI) has been proved to be an efficient solution to extend the communication range. Due to the small coil antenna's physical limitation, however, MI's communication range is still very limited. To this end, Metamaterial-enhanced Magnetic Induction (M$^2$I) communication has been proposed and the theoretical results suggest that it can significantly increase the communication performance, namely, data rate and communication range. Nevertheless, currently, the real implementation of M$^2$I is still a challenge and there is no guideline on design and fabrication of spherical metamaterial. In this paper, we propose a practical design by using a spherical coil array to realize M$^2$I and we prove that it can achieve negative permeability and there exists a resonance condition where the radiated magnetic field can be significantly amplified. The radiation and communication performance are ev...

  19. 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.

  20. M2-F1 on lakebed with pilot Milt Thompson

    Science.gov (United States)

    1963-01-01

    NASA Flight Research Pilot Milt Thompson, shown here on the lakebed with the M2-F1 lifting body, was an early backer of R. Dale Reed's lifting-body proposal. He urged Flight Research Center director Paul Bikle to approve the M2-F1's construction. Thompson also made the first glide flights in both the M2-F1 and its successor, the heavyweight M2-F2. 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, NASA Flight Research Center (later Dryden Flight Research Center, Edwards, CA) 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

  1. Integrated Self-Assembly of the Mms6 Magnetosome Protein to Form an Iron-Responsive Structure

    Directory of Open Access Journals (Sweden)

    Marit Nilsen-Hamilton

    2013-07-01

    Full Text Available A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe3+. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding.

  2. CEMS-investigations of AISI M2 steel after nitrogen plasma immersion ion implantation

    Energy Technology Data Exchange (ETDEWEB)

    Uglov, V.V. E-mail: info@research.bsu.unibel.by; Kholmetskii, A.L.; Kuleshov, A.K.; Fedotova, J.A.; Rusalsky, D.P.; Khodasevich, V.V.; Ruebenbauer, K.; Richter, E.; Guenzel, R.; Parascandola, S

    1999-01-02

    Plasma-immersion ion implantation (PIII) and high-current ion implantation (HCII) of nitrogen into M2 steel were carried out. The formation of a multilayer system consisting of a surface layer of austenite on top of {epsilon}-Fe{sub 3}N nitride precipitates was manifested in M2 steel after PIII. HCII forms a thick (more than 14 {mu}m) layer with {epsilon}-Fe{sub 3}N precipitates. Apparently, the formation of the austenite layer after PIII is connected with a local tempering of the steel surface by the pulsed nitrogen ion beam.

  3. M2 macrophages induce EMT through the TGF-β/Smad2 signaling pathway.

    Science.gov (United States)

    Zhu, Liangying; Fu, Xiao; Chen, Xiang; Han, Xiaodong; Dong, Ping

    2017-09-01

    IPF is characterized by fibroblast accumulation, collagen deposition, and ECM remodeling, with myofibroblasts believed to be the effector cell type. Myofibroblasts develop due to EMT of lung alveolar epithelial cells, which can be induced by TGF-β. M2 macrophages, a macrophage subpopulation, secrete large amounts of TGF-β. To clarify the relationship between IPF, EMT, TGF-β, and M2 macrophages, a bleomycin-induced pulmonary fibrosis mouse model was used. Seventeen days after mice were treated with bleomycin, the successful establishment of a pulmonary fibrosis model was confirmed by HE stain and Masson's trichrome stain. We found evidence in support of EMT, such as elevated protein levels of α-SMA in lung tissue and decreased levels of E-cadherin and CK-18. Additionally, increased TGF-β levels and TGF-β/Smad2 signaling activation was observed. Macrophages were recruited to pulmonary alveoli. Alveolar macrophages were phenotyped and identified as M2 macrophages, with up-regulated CD206 on the cell surfaces. For in vitro studies, we treated RAW 264.7 cells with IL-4 for 24 h, and the cells were then utilized as M2 macrophages. TGF-β levels increased significantly in the culture supernatant. Forty-eight hours after lung epithelial cells (MLE-12) were co-cultured with the M2 macrophages, the expression of α-SMA increased, and E-cadherin and CK-18 decreased. When a TGF-β receptor inhibitor, LY2109761 was used, the EMT induced by M2 macrophages was blocked. In conclusion, we demonstrated that M2 macrophages induce EMT through the TGF-β/Smad2 signaling pathway. © 2017 International Federation for Cell Biology.

  4. Thermal Decomposition of Anhydrous Alkali Metal Dodecaborates M2B12H12 (M = Li, Na, K

    Directory of Open Access Journals (Sweden)

    Liqing He

    2015-11-01

    Full Text Available Metal dodecaborates M2/nB12H12 are regarded as the dehydrogenation intermediates of metal borohydrides M(BH4n that are expected to be high density hydrogen storage materials. In this work, thermal decomposition processes of anhydrous alkali metal dodecaborates M2B12H12 (M = Li, Na, K synthesized by sintering of MBH4 (M = Li, Na, K and B10H14 have been systematically investigated in order to understand its role in the dehydrogenation of M(BH4n. Thermal decomposition of M2B12H12 indicates multistep pathways accompanying the formation of H-deficient monomers M2B12H12−x containing the icosahedral B12 skeletons and is followed by the formation of (M2B12Hzn polymers. The decomposition behaviors are different with the in situ formed M2B12H12 during the dehydrogenation of metal borohydrides.

  5. Electrophoretic and spectroscopic characterization of the protein patterns formed in different surfactant solutions.

    Science.gov (United States)

    Blanco, Elena; Ruso, Juan M; Prieto, Gerardo; Sarmiento, Félix

    2008-01-01

    The complexations between catalase and the sodium perfluorooctanoate/sodium octanoate and sodium perfluorooctanoate/sodium dodecanoate systems have been studied by a combination of electrophoresis and spectroscopy measurements. The numbers of adsorption sites on the protein were determined from the observed increases of the zeta-potential as a function of surfactant concentration in the regions where the adsorption was a consequence of the hydrophobic effect. The Gibbs energies of adsorption of the surfactants onto the protein were evaluated and the results show that for all systems, Gibbs energies are negative and larger, in absolute values, at low values of surfactant concentration where binding to the high energy sites takes place, and become less negative as more surfactant molecules bind, suggesting a saturation process. The role of hydrophobic interactions in these systems has been demonstrated to be the predominant. Spectroscopy measurements suggest conformational changes on catalase depending on the surfactant mixture as well as the mixed ratio. No isosbestic point or shifts have been found showing that catalase has spectrophotometrically one kind of binding site for these surfactant mixtures.

  6. Deposition of Bacteriorhodopsin Protein in a Purple Membrane Form on Nitrocellulose Membranes for Enhanced Photoelectric Response

    Directory of Open Access Journals (Sweden)

    Chang-Hoon Nam

    2012-12-01

    Full Text Available Bacteriorhodopsin protein (bR-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

  7. Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

    Science.gov (United States)

    Kim, Young Jun; Neuzil, Pavel; Nam, Chang-Hoon; Engelhard, Martin

    2012-12-27

    Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

  8. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

    Science.gov (United States)

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

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

    Science.gov (United States)

    Gilbert, Leona; Toivola, Jouni; Välilehto, Outi; Saloniemi, Taija; Cunningham, Claire; White, Daniel; Mäkelä, Anna R; Korhonen, Eila; Vuento, Matti; Oker-Blom, Christian

    2006-12-08

    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.

  10. 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.

  11. M2磁带机挑战极限

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    日前安百特(Exabyte)公司宣布,其OEM合作伙伴Cybernetics在对Mammoth-2(M2)磁带机的性能和可靠性测试中取得了创记录的结果,从而加快了M2的商业交付计划。M2磁带机以部门级价格提供了企业级的性能,其容量增加50%,达到60GB,性能提高一倍,传输率达到12MB/s,可在一小时内备份43GB数据(未压缩)。Cybernetics利用该公司为了故意“破坏”磁带机而创建的定制软件对M2进行了测试,包括读/写能力测试、

  12. 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 tra

  13. Progress On 58m2 Passive Resonant Ring Laser Gyroscope

    Science.gov (United States)

    Shaw, G. L.; Rotge, J.; Simmons, B. J.

    1986-01-01

    An update of the large area (now 60m2) Passive Resonant Ring Laser Gyro (PRRLG) is given. Some aspects of last year's design have changed; but performance is still predicted to be in the 10-10 earth rate unit (ERU) range. This is of interest for a number of geophysical applications.

  14. Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A▿

    Science.gov (United States)

    Garibal, Julie; Hollville, Émilie; Bell, Andrew I.; Kelly, Gemma L.; Renouf, Benjamin; Kawaguchi, Yasushi; Rickinson, Alan B.; Wiels, Joëlle

    2007-01-01

    The Epstein-Barr virus (EBV)-encoded leader protein, EBNA-LP, strongly activates the EBNA2-mediated transcriptional activation of cellular and viral genes and is therefore important for EBV-induced B-cell transformation. However, a truncated form of EBNA-LP is produced in cells infected with variant EBV strains lacking EBNA2 due to a genetic deletion. The function of this truncated form is unknown. We show here that some Burkitt's lymphoma cells harboring defective EBV strains are specifically resistant to the caspase-dependent apoptosis induced by verotoxin 1 (VT-1) or staurosporine. These cells produced low-molecular-weight Y1Y2-truncated isoforms of EBNA-LP, which were partly localized in the cytoplasm. The transfection of sensitive cells with constructs encoding truncated EBNA-LP isoforms, but not full-length EBNA-LP, induced resistance to caspase-mediated apoptosis. Furthermore, VT-1 induced protein phosphatase 2A (PP2A) activation in sensitive cells but not in resistant cells, in which the truncated EBNA-LP interacted with this protein. Thus, the resistance to apoptosis observed in cells harboring defective EBV strains most probably results from the inactivation of PP2A via interactions with low-molecular-weight Y1Y2-truncated EBNA-LP isoforms. PMID:17494066

  15. A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

    Science.gov (United States)

    Al-Nihmi, Faisal Mohammed Abdul; Kolli, Surendra Kumar; Reddy, Segireddy Rameswara; Mastan, Babu S.; Togiri, Jyothi; Maruthi, Mulaka; Gupta, Roshni; Sijwali, Puran Singh; Mishra, Satish; Kumar, Kota Arun

    2017-01-01

    Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus. PMID:28067322

  16. Internal steel structure of M2-F1

    Science.gov (United States)

    1963-01-01

    The internal steel structure for the M2-F1 was built at the Flight Research Center (predecessor of the Dryden Flight Research Center, Edwards, CA) in a section of the calibration hangar dubbed 'Wright Bicycle Shop.' Visible are the stick, rudder pedals, and ejection seat. The external wooden shell was attached to the steel structure. 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

  17. Sperm Lysozyme‐Like Protein 1 (SLLP1), an intra‐acrosomal oolemmal‐binding sperm protein, reveals filamentous organization in protein crystal form

    National Research Council Canada - National Science Library

    Zheng, H; Mandal, A; Shumilin, I. A; Chordia, M. D; Panneerdoss, S; Herr, J. C; Minor, W

    2015-01-01

    .... mSLLP 1 is distinct from c‐lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75...

  18. The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein.

    Science.gov (United States)

    Brázda, Václav; Čechová, Jana; Battistin, Michele; Coufal, Jan; Jagelská, Eva B; Raimondi, Ivan; Inga, Alberto

    2017-01-29

    The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.

  19. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    Science.gov (United States)

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  20. Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

    Science.gov (United States)

    Schweighofer, K. J.; Pohorille, A.

    2000-01-01

    The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).

  1. The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Melanie F Kho

    Full Text Available The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry proteins, which are pore-forming toxins or pore-forming proteins (PFPs. Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.

  2. Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

    Science.gov (United States)

    Shibata, Y; Okano, S; Shiroza, T; Tahara, T; Nakazawa, K; Kataoka, S; Ishida, I; Kobayashi, T; Yoshie, H; Abiko, Y

    2011-12-01

    The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth. © 2011 John Wiley & Sons A/S.

  3. 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.

  4. The secreted form of the Alzheimer's beta-amyloid precursor protein stimulates a membrane-associated guanylate cyclase.

    OpenAIRE

    Barger, S. W.; Mattson, M.P.

    1995-01-01

    We previously demonstrated that secreted forms of the Alzheimer's beta-amyloid precursor protein (sAPP) elevate cyclic GMP (cGMP) in primary neuronal cultures and that this effect is responsible for the modulation of neuronal calcium homoeostasis by sAPP. We have investigated further the mechanism by which sAPP elevates cGMP. Inhibition of the formation of nitric oxide or carbon monoxide did not affect the ability of sAPP to lower rapidly intraneuronal calcium levels or elevate cGMP, suggesti...

  5. Distribution between protein-bound and free forms of plasma cortisol in the gilt and fetal pig near term.

    Science.gov (United States)

    Kattesh, H G; Baumbach, G A; Gillespie, B B; Schneider, J F; Murai, J T

    1997-01-01

    Thirty-five time-dated pregnant gilts were used to document plasma levels of total and free cortisol, corticosteroid-binding globulin (CBG) binding capacity, and percent distribution of cortisol among protein-bound (CBG and albumin) and free forms in the fetal pig during the last 24 days of gestation. Plasma from fetal pigs on days 110-114 of gestation (gestation length 114 days) had significantly higher levels of total cortisol (p pigs located in the cervical region of the uterus had lower (p pig are directly related and highly similar to those of another precocious species, the sheep.

  6. Postsynaptic protein synthesis is required for presynaptic enhancement in persistent forms of long-term potentiation.

    Directory of Open Access Journals (Sweden)

    Victoria Philippa Anne Johnstone

    2013-02-01

    Full Text Available Long-term potentiation (LTP in the hippocampus is a fundamental process underlying learning and memory in the brain. At CA3-CA1 synapses, three discrete forms of LTP (LTP1, 2 and 3 have been differentiated on the basis of their persistence, maintenance mechanisms, Ca2+ signaling pathways, expression loci, and electrophysiological requirements. We previously showed that LTP2 and LTP3 involve a presynaptic expression component that is established in a translation-dependent manner. Here we investigate the locus of translation required for presynaptic expression. Neurotransmitter release rate was estimated via FM 1-43 destaining from CA3 terminals in hippocampal slices from male Wistar rats (6-8 weeks. Destaining was measured at sites making putative contact with CA1 dendritic processes in stratum radiatum that had been filled with a membrane impermeable translation inhibitor and a fluorescent indicator. Our results suggest that inhibition of postsynaptic translation eliminates the enhanced release ordinarily observed at 160 min post LTP induction, and that this effect is limited to sites closely apposed to the filled postsynaptic cell. We conclude that postsynaptic translation is required for the presynaptic component of LTP2 and LTP3 expression. These data considerably strengthen the mechanistic separation of LTP1, 2 and 3 and provide evidence for an expanded repertoire of communication between synaptic elements.

  7. Islet amyloid polypeptide forms rigid lipid-protein amyloid fibrils on supported phospholipid bilayers.

    Science.gov (United States)

    Domanov, Yegor A; Kinnunen, Paavo K J

    2008-02-08

    Islet amyloid polypeptide (IAPP) forms fibrillar amyloid deposits in the pancreatic islets of Langerhans of patients with type 2 diabetes mellitus, and its misfolding and aggregation are thought to contribute to beta-cell death. Increasing evidence suggests that IAPP fibrillization is strongly influenced by lipid membranes and, vice versa, that the membrane architecture and integrity are severely affected by amyloid growth. Here, we report direct fluorescence microscopic observations of the morphological transformations accompanying IAPP fibrillization on the surface of supported lipid membranes. Within minutes of application in submicromolar concentrations, IAPP caused extensive remodeling of the membrane including formation of defects, vesiculation, and tubulation. The effects of IAPP concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. Growth of amyloid fibrils was visualized using fluorescently labeled IAPP or thioflavin T staining. Two-color imaging of the peptide and membranes revealed that the fibrils were initially composed of the peptide only, and vesiculation occurred in the points where growing fibers touched the lipid membrane. Interestingly, after 2-5 h of incubation, IAPP fibers became "wrapped" by lipid membranes derived from the supported membrane. Progressive increase in molecular-level association between amyloid and membranes in the maturing fibers was confirmed by Förster resonance energy transfer spectroscopy.

  8. The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis.

    Science.gov (United States)

    McGlinchey, Ryan P; Shewmaker, Frank; McPhie, Peter; Monterroso, Begoña; Thurber, Kent; Wickner, Reed B

    2009-08-18

    Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

  9. Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

    Science.gov (United States)

    Yang, Yi; Wikieł, Agata J; Dall'Agnol, Leonardo T; Eloy, Pierre; Genet, Michel J; Moura, José J G; Sand, Wolfgang; Dupont-Gillain, Christine C; Rouxhet, Paul G

    2016-01-01

    The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

  10. Dissociated GαGTP and Gβγ protein subunits are the major activated form of heterotrimeric Gi/o proteins.

    Science.gov (United States)

    Bondar, Alexey; Lazar, Josef

    2014-01-17

    Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.

  11. Wilson Loops for M2- and M5-brane spaces

    CERN Document Server

    Quijada, Edward

    2015-01-01

    We calculate the quark and anti-quark interaction energy in different positions in spaces generated by $N$ coincident $M2$- and $M5$-branes. We use the Maldacena-Rey-Yee method for calculating this energy as a function of quark-antiquark separation. We obtain the solution for these problems as integrals of the metric elements. For limiting regimes we find simpler solutions for which some potentials exhibit a confinement behavior.

  12. Tumor-derived interleukin-1 promotes lymphangiogenesis and lymph node metastasis through M2-type macrophages.

    Directory of Open Access Journals (Sweden)

    Kosuke Watari

    Full Text Available Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF-C through its receptor (VEGFR-3 and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.

  13. Tumor-derived interleukin-1 promotes lymphangiogenesis and lymph node metastasis through M2-type macrophages.

    Science.gov (United States)

    Watari, Kosuke; Shibata, Tomohiro; Kawahara, Akihiko; Sata, Ken-ichi; Nabeshima, Hiroshi; Shinoda, Ai; Abe, Hideyuki; Azuma, Koichi; Murakami, Yuichi; Izumi, Hiroto; Takahashi, Takashi; Kage, Masayoshi; Kuwano, Michihiko; Ono, Mayumi

    2014-01-01

    Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.

  14. Membrane damage by an α-helical pore-forming protein, Equinatoxin II, proceeds through a succession of ordered steps.

    Science.gov (United States)

    Rojko, Nejc; Kristan, Katarina Č; Viero, Gabriella; Žerovnik, Eva; Maček, Peter; Dalla Serra, Mauro; Anderluh, Gregor

    2013-08-16

    Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.

  15. Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action.

    Science.gov (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-12-15

    The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.

  16. 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.

  17. Protection against Multiple Influenza A Virus Strains Induced by Candidate Recombinant Vaccine Based on Heterologous M2e Peptides Linked to Flagellin

    Science.gov (United States)

    Kovaleva, Anna A.; Potapchuk, Marina V.; Korotkov, Alexandr V.; Sergeeva, Mariia V.; Kasianenko, Marina A.; Kuprianov, Victor V.; Ravin, Nikolai V.; Tsybalova, Liudmila M.; Skryabin, Konstantin G.; Kiselev, Oleg I.

    2015-01-01

    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. PMID:25799221

  18. 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 pho

  19. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    Science.gov (United States)

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    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.

  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. Notch-induced Asb2 expression promotes protein ubiquitination by forming non-canonical E3 ligase complexes

    Institute of Scientific and Technical Information of China (English)

    Lei Nie; Ying Zhao; Wei Wu; Yuan-Zheng Yang; Hong-Cheng Wang; Xiao-Hong Sun

    2011-01-01

    Notch signaling controls multiple developmental processes, thus demanding versatile functions. We have previously shown that this may be partly achieved by accelerating ubiquitin-mediated degradation of important regulators of differentiation. However, the underlying mechanism was unknown. We now find that Notch signaling transcriptionally activates the gene encoding ankyrin-repeat SOCS box-containing protein 2(Asb2). Asb2 promotes the ubiquidnation of Notch targets such as E2A and Janus kinase(Jak)2, and a dominant-negative(DN)mutant of Asb2blocks Notch-induced degradation of these proteins. Asb2 likely binds Jak2 directly but associates with E2A through Skp2. We next provide evidence to suggest that Asb2 bridges the formation of non-canonical cullin-based complexes through interaction with not only ElonginB/C and Cullin(Cul)5, but also the F-box-containing protein, Skp2, which is known to associate with Skpl and Cull. Consistently, ablating the function of Cull or Cu15 using DN mutants or siRNAs protected both E2A and Jak2 from Asb2-mediated or Notch-induced degradation. By shifting monomeric E3ligase complexes to dimeric forms through activation of Asb2 transcription, Notch could effectively control the turnover of a variety of substrates and it exerts diverse effects on cell proliferation and differentiation.

  2. 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.

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

    Science.gov (United States)

    Liu, Angela M; Xu, Zhi; Shek, Felix H; Wong, Kwong-Fai; Lee, Nikki P; Poon, Ronnie T; Chen, Jinfei; Luk, John M

    2014-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Qing; Wang; Mei-Lan; Xue; Gui-Qiu; Zhao; Mei-Guang; Liu; Yu-Na; Ma; Yan; Ma

    2015-01-01

    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 m RNAs for BMP-2,-4,-5,-7,-RIA,-RIB and BMP-RII. Conversely, rat sclera only expressed m RNA 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(P <0.05 vs internal control eyes).· CONCLUSION: 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. 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.

  6. ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

    Science.gov (United States)

    Hui, Monica P; Galkin, Vitold E; Yu, Xiong; Stasiak, Alicja Z; Stasiak, Andrzej; Waldor, Matthew K; Egelman, Edward H

    2010-03-01

    Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

  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. 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.

  9. M2-branes and AdS/CFT

    CERN Document Server

    Klebanov, Igor R

    2009-01-01

    These notes provide a brief introduction to the ABJM theory, the level k U(N) x U(N) superconformal Chern-Simons matter theory which has been conjectured to describe N coincident M2-branes. We discuss its dual formulation in terms of M-theory on AdS_4 x S^7/Z_k and review some of the evidence in favor of the conjecture. We end with a brief discussion of the important role played by the monopole operators.

  10. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.

    Science.gov (United States)

    Tabita, F Robert; Satagopan, Sriram; Hanson, Thomas E; Kreel, Nathan E; Scott, Stephanie S

    2008-01-01

    There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

  11. Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments.

    Science.gov (United States)

    Bera, Amal K; Akabas, Myles H

    2005-10-21

    The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

  12. A comprehensive survey of M(2)AX phase elastic properties.

    Science.gov (United States)

    Cover, M F; Warschkow, O; Bilek, M M M; McKenzie, D R

    2009-07-29

    M(2)AX phases are a family of nanolaminate, ternary alloys that are composed of slabs of transition metal carbide or nitride (M(2)X) separated by single atomic layers of a main group element. In this combination, they manifest many of the beneficial properties of both ceramic and metallic compounds, making them attractive for many technological applications. We report here the results of a large scale computational survey of the elastic properties of all 240 elemental combinations using first-principles density functional theory calculations. We found correlations revealing the governing role of the A element and its interaction with the M element on the c axis compressibility and shearability of the material. The role of the X element is relatively minor, with the strongest effect seen in the in-plane constants C(11) and C(12). We identify several elemental compositions with extremal properties such as W(2)SnC, which has by far the lowest value of C(44), suggesting potential applications as a high-temperature dry lubricant.

  13. 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.

  14. Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hua Jiang; Ren-Qian Zhong; Sheng-Qian Yu; Yin Hu; Weng-Weng Li; Xian-Tao Kong

    2003-01-01

    AIM: To construct and express a humanized M2 autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC). METHODS: cDNA fragments encoding M2-reactive epitopes of pyruvate dehydrogenase complex Ez (PDCE2), branched chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) and 2-oxo-glutarate dehydrogenase complex E2 (OGDC-E2) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into the plasmid vector pQE-30 and then transferred into E. coliM15 (pREP4) for expression, which was induced by isopropylthio-β-Dgalactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M2-positive sera confirmed at Euroimmun Research Center (Germany).Using the purified BPO, M2 antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA. RESULTS: The expressed BPO was observed with both antigenic reactivity and specificity of M2 autoantigens. The determination of M2 antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay.With the newly established method, M2 antibodies were found in 100 % (20/20) of patients with PBC. Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M2antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M2-antibody positive. CONCLUSION: Compared with the routine immunofluorescenoe assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.

  15. Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.

    Science.gov (United States)

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J; Robinson, Colin

    2012-03-01

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.

  16. 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.

  17. Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

    Science.gov (United States)

    Hilbert, Manuel; Erat, Michèle C; Hachet, Virginie; Guichard, Paul; Blank, Iris D; Flückiger, Isabelle; Slater, Leanne; Lowe, Edward D; Hatzopoulos, Georgios N; Steinmetz, Michel O; Gönczy, Pierre; Vakonakis, Ioannis

    2013-07-09

    Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.

  18. 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.

  19. Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns.

    Science.gov (United States)

    Chan, Yen-Hui; Huang, Tsung-Wei; Young, Tai-Horng; Lou, Pei-Jen

    2011-11-01

    Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.

  20. Protein kinase B (PKB/AKT1) formed signaling complexes with mitochondrial proteins and prevented glycolytic energy dysfunction in cultured cardiomyocytes during ischemia-reperfusion injury.

    Science.gov (United States)

    Deng, Wu; Leu, Hsin-Bang; Chen, Yumay; Chen, Yu-Han; Epperson, Christine M; Juang, Charity; Wang, Ping H

    2014-05-01

    Our previous studies showed that insulin stimulated AKT1 translocation into mitochondria and modulated oxidative phosphorylation complex V in cardiac muscle. This raised the possibility that mitochondrial AKT1 may regulate glycolytic oxidative phosphorylation and mitochondrial function in cardiac muscle cells. The aims of this project were to study the effects of mitochondrial AKT1 signaling on cell survival in stressed cardiomyocytes, to define the effect of mitochondrial AKT1 signaling on glycolytic bioenergetics, and to identify mitochondrial targets of AKT1 signaling in cardiomyocytes. Mitochondrial AKT1 signaling played a protective role against apoptosis and necrosis during ischemia-reperfusion stress, suppressed mitochondrial calcium overload, and alleviated mitochondrial membrane depolarization. Activation of AKT1 signaling in mitochondria increased glucose uptake, enhanced respiration efficiency, reduced superoxide generation, and increased ATP production in the cardiomyocytes. Inhibition of mitochondrial AKT attenuated insulin response, indicating that insulin regulation of ATP production required mitochondrial AKT1 signaling. A proteomic approach was used to reveal 15 novel targets of AKT1 signaling in mitochondria, including pyruvate dehydrogenase complex (PDC). We have confirmed and characterized the association of AKT1 and PDC subunits and verified a stimulatory effect of mitochondrial AKT1 on the enzymatic activity of PDC. These findings suggested that AKT1 formed protein complexes with multiple mitochondrial proteins and improved mitochondrial function in stressed cardiomyocytes. The novel AKT1 signaling targets in mitochondria may become a resource for future metabolism research.

  1. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    Science.gov (United States)

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  2. 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.

  3. Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus

    Energy Technology Data Exchange (ETDEWEB)

    Love, Robert A.; Brodsky, Oleg; Hickey, Michael J.; Wells, Peter A.; Cronin, Ciarán N.; Pfizer

    2009-07-10

    A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

  4. D6 PROTEIN KINASE activates auxin transport-dependent growth and PIN-FORMED phosphorylation at the plasma membrane.

    Science.gov (United States)

    Barbosa, Inês C R; Zourelidou, Melina; Willige, Björn C; Weller, Benjamin; Schwechheimer, Claus

    2014-06-23

    The directed cell-to-cell transport of the phytohormone auxin by efflux and influx transporters is essential for proper plant growth and development. Like auxin efflux facilitators of the PIN-FORMED (PIN) family, D6 PROTEIN KINASE (D6PK) from Arabidopsis thaliana localizes to the basal plasma membrane of many cells, and evidence exists that D6PK may directly phosphorylate PINs. We find that D6PK is a membrane-bound protein that is associated with either the basal domain of the plasma membrane or endomembranes. Inhibition of the trafficking regulator GNOM leads to a rapid internalization of D6PK to endomembranes. Interestingly, the dissociation of D6PK from the plasma membrane is also promoted by auxin. Surprisingly, we find that auxin transport-dependent tropic responses are critically and reversibly controlled by D6PK and D6PK-dependent PIN phosphorylation at the plasma membrane. We conclude that D6PK abundance at the plasma membrane and likely D6PK-dependent PIN phosphorylation are prerequisites for PIN-mediated auxin transport.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Carina S S Gomes-Santos

    2011-05-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Carina S S Gomes-Santos

    2011-05-01

    Full Text Available 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.

  8. Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿

    Science.gov (United States)

    Nogueira, Sarah Veloso; Fonseca, Fernanda L.; Rodrigues, Marcio L.; Mundodi, Vasanth; Abi-Chacra, Erika A.; Winters, Michael S.; Alderete, John F.; Soares, Célia Maria de Almeida

    2010-01-01

    Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis. PMID

  9. Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells.

    Science.gov (United States)

    Nogueira, Sarah Veloso; Fonseca, Fernanda L; Rodrigues, Marcio L; Mundodi, Vasanth; Abi-Chacra, Erika A; Winters, Michael S; Alderete, John F; de Almeida Soares, Célia Maria

    2010-09-01

    Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

  10. TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

    Science.gov (United States)

    Skopin, Anton; Shalygin, Alexey; Vigont, Vladimir; Zimina, Olga; Glushankova, Lyubov; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2013-02-01

    TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

  11. Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts.

    Science.gov (United States)

    Machuca-Tzili, Laura; Thorpe, Helena; Robinson, Thelma E; Sewry, Caroline; Brook, J David

    2006-11-01

    Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.

  12. Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.

    Science.gov (United States)

    Chittibabu, G; Ma, Charles; Netter, Hans J; Noronha, Santosh B; Coppel, Ross L

    2014-04-01

    Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

  13. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

    Directory of Open Access Journals (Sweden)

    Larry J Bischof

    2008-10-01

    Full Text Available Pore-forming toxins (PFTs constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

  14. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

    Directory of Open Access Journals (Sweden)

    Larry J Bischof

    2008-10-01

    Full Text Available Pore-forming toxins (PFTs constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

  15. t-Darpp stimulates protein kinase A activity by forming a complex with its RI regulatory subunit.

    Science.gov (United States)

    Theile, Dirk; Geng, Shuhui; Denny, Erin C; Momand, Jamil; Kane, Susan E

    2017-09-01

    t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDp(T39A)), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDp(T39A) cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp-mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process.

    Science.gov (United States)

    Hapuarachchi, Suminda; Fodor, Szilan; Apostol, Izydor; Huang, Gang

    2011-07-15

    Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.

  17. 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.

  18. Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

    Science.gov (United States)

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Nomura, Norimichi

    2014-12-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

  19. The Extreme Type I Planetary Nebula M2-52

    Directory of Open Access Journals (Sweden)

    M. Peña

    2002-01-01

    Full Text Available Se presentan los resultados obtenidos a partir de espectroscopía de alta resolución de la parte central de la nebulosa planetaria bipolar M2-52 que muestra un tipo morfológico Br. Hemos confirmado que M2-52 es una nebulosa de Tipo I de Peimbert, con un espectro rico en líneas de alto y bajo grado de ionización y un fuerte enriquecimiento de He y N. La composición química del gas ionizado es: He/H = 0:165 0:010, O/H = (2:6 0:5 x 10_4, N/O = 2:3 0:3, Ne/O = 0:37 0:10, Ar/O = (9:2 2:0 x 10_3 y S/O > 2:0 x 10_3. La velocidad de expansión de la nebulosa es, en promedio, de 20 2 km s_1 y varía ligeramente dependiendo del ión considerado. Los iones de menor grado de ionización, N+ y S+, muestran vexp _ 18 km s_1, O++ y He+ muestran vexp _ 20 km s_1, en tanto que He++ y H+ muestran vexp _ 22 km s_1. Es posible que la zona de N+ y S+ esté siendo frenada por el anillo de material molecular encontrado alrededor de la estrella.

  20. Marginal fluctuations as instantons on M2/D2-branes

    Energy Technology Data Exchange (ETDEWEB)

    Naghdi, M. [University of Ilam, Department of Physics, Faculty of Basic Sciences, Ilam (Iran, Islamic Republic of)

    2014-03-15

    We introduce some (anti-) M/D-branes through turning on the corresponding field strengths of the 11- and 10-dimensional supergravity theories over AdS{sub 4} x M{sup 7} {sup vertical} {sup stroke} {sup 6} spaces, where we use S{sup 7}/Z{sub k} and CP{sup 3} for the internal spaces. Indeed, when we add M2/D2-branes on the same directions with the near horizon branes of the Aharony-Bergman-Jafferis- Maldacena model, all symmetries and supersymmetries are preserved trivially. In this case, we obtain a localized object just in the horizon. This normalizable bulk massless scalar mode is a singlet of SO(8) and SU(4) x U(1), and it agrees with a marginal boundary operator of the conformal dimension of Δ{sub +} = 3. However, after performing a special conformal transformation, we see that the solution is localized in the Euclideanized AdS{sub 4} space and is attributable to the included anti-M2/D2-branes, which are also necessary to ensure that there is no back-reaction. The resultant theory now breaks all N = 8, 6 supersymmetries to N = 0, while the other symmetries are so preserved. The dual boundary operator is then set up from the skew-whiffing of the representations 8s and 8v for the supercharges and scalars, respectively, while the fermions remain fixed in 8c of the original theory. Besides, we also address another alternate bulk to boundary matching procedure through turning on one of the gauge fields of the full U(N){sub k} x U(N){sub -k} gauge group along the same lines with a similar situation to the one faced in the AdS{sub 5}/CFT{sub 4} correspondence. The latter approach covers the difficulty already faced with in the bulk-boundary matching procedure for k = 1, 2 as well. (orig.)

  1. The soluble periplasmic domains of Escherichia coli cell division proteins FtsQ/FtsB/FtsL form a trimeric complex with submicromolar affinity

    NARCIS (Netherlands)

    Glas, Marjolein; Bart Van Den Berg Van Saparoea, H.; McLaughlin, Stephen H.; Roseboom, Winfried; Liu, Fan; Koningstein, Gregory M.; Fish, Alexander; Den Blaauwen, Tanneke; Heck, Albert J R; De Jong, Luitzen; Bitter, Wilbert; De Esch, Iwan J P; Luirink, Joen

    2015-01-01

    Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a ce

  2. The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL form a Trimeric Complex with Sub-micromolar Affinity

    NARCIS (Netherlands)

    Glas, M.; van den Berg van Saparoea, H.B.; McLaughlin, S.H.; Roseboom, W.; Liu, F.; Koningstein, G.M.; Fish, A.; den Blaauwen, T.; Heck, A.J.R.; de Jong, L.; Bitter, W.; de Esch, I.J.P.; Luirink, J.

    2015-01-01

    Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a ce

  3. Carboxyl Terminus Truncated Human Papillomavirus Type 58 L1 Protein Maintains Its Bioactivity and Ability to Form Virus-like Particles

    Institute of Scientific and Technical Information of China (English)

    李文生; 刘红莉; 郑瑾; 陈宏伟; 杨军; 王丽秀; 闫小飞; 王一理; 司履生

    2004-01-01

    To prepare carboxyl terminus truncated human papillomavirus type 58 L1 (HPV58L1)protein and evaluate its ability to form virusqike particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing - HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBondTM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope.Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

  4. Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q.

    Science.gov (United States)

    Moreau, Christophe; Bally, Isabelle; Chouquet, Anne; Bottazzi, Barbara; Ghebrehiwet, Berhane; Gaboriaud, Christine; Thielens, Nicole

    2016-01-01

    Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A-C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed.

  5. Unveiling the structure of the planetary nebula M 2-48 Kinematics and physical conditions

    CERN Document Server

    López-Martin, L; Esteban, C; Vázquez, R; Raga, A C; Torrelles, J M; Miranda, L F; Meaburn, J; Olguin, L

    2002-01-01

    The kinematics and physical conditions of the bipolar planetary nebula M 2-48 are analysed from high and low dispersion long-slit spectra. Previous CCD narrow-band optical observations have suggested that this nebula is mainly formed by a pair of symmetric bow-shocks, an off-center semi-circular shell, and an internal bipolar structure. The bipolar outflow has a complex structure, characterised by a series of shocked regions located between the bright core and the polar tips. There is an apparent kinematic discontinuity between the bright bipolar core and the outer regions. The fragmented ring around the bright bipolar region presents a low expansion velocity and could be associated to ejection in the AGB-PN transition phase, although its nature remains unclear. The chemical abundances of the central region are derived, showing that M 2-48 is a Type I planetary nebula (PN).

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

    Science.gov (United States)

    Bi, Jia; Zeng, Xianxin; Zhao, Lin; Wei, Qian; Yu, Lifeng; Wang, Xinnan; Yu, Zhaojin; Cao, Yaming; Shan, Fengping; Wei, Minjie

    2016-01-01

    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. PMID:27673564

  7. Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H

    1980-01-01

    revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed...... immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form...... in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar...

  8. Down-regulated expression of NPM1 in IMS-M2 cell line by (-)-epigallocatechin-3-gallate

    Institute of Scientific and Technical Information of China (English)

    Hoang Thanh Chi; Bui Thi Kim Ly; Hoang Anh Vu; Yuko Sato; Phu Chi Dung; Phan Thi Xinh

    2014-01-01

    Objective: To investigate the inhibited effect of epigallocatechin-3-gallate (EGCG) on the expression of NPM1 in IMS-M2 cells harboring the NPM1 mutations.Methods:Cell proliferation assay was performed to test the effects of EGCG on cell growth of IMS-M2 cells harboring the NPM1 mutations. Western blot analysis were performed to test the protein expression of NPM1, AKT, those associated with apoptosis. Results: EGCG can down-regulate the expression of NPM1 in IMS-M2 cells harboring the NPM1 mutations. Moreover, EGCG also suppressed the cell proliferation and induced apoptosis in IMS-M2 cells.Conclusions:The results suggested that EGCG could be considered as a reagent for treatment of AML patients with NPM1 mutations.

  9. Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice.

    Science.gov (United States)

    Rappazzo, C Garrett; Watkins, Hannah C; Guarino, Cassandra M; Chau, Annie; Lopez, Jody L; DeLisa, Matthew P; Leifer, Cynthia A; Whittaker, Gary R; Putnam, David

    2016-03-04

    Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. 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.

  11. Multiwavelength photometry in the Globular Cluster M2

    CERN Document Server

    Dalessandro, E; Lanzoni, B; Ferraro, F R; Schiavon, R; Rood, R T

    2009-01-01

    We present a multiwavelength photometric analysis of the globular cluster M2. The data-set has been obtained by combining high-resolution (HST/WFPC2 and ACS) and wide-field (GALEX) space observations and ground based (MEGACAM-CFHT, EMMI-NTT) images. The photometric sample covers the entire cluster extension from the very central regions up to the tidal radius and beyond. It allows an accurate determination of the cluster center of gravity and other structural parameters derived from the star count density profile. Moreover we study the BSS population and its radial distribution. A total of 123 BSS has been selected, and their radial distribution has been found to be bimodal (highly peaked in the center, decreasing at intermediate radii and rising outward), as already found in a number of other clusters. The radial position of the minimum of the BSS distribution is consistent with the radius of avoidance caused by the dynamical friction of massive objects over the cluster age. We also searched for gradients in...

  12. Metric 3-Leibniz algebras and M2-branes

    CERN Document Server

    Méndez-Escobar, Elena

    2010-01-01

    This thesis is concerned with superconformal Chern-Simons theories with matter in 3 dimensions. The interest in these theories is two-fold. On the one hand, it is a new family of theories in which to test the AdS/CFT correspondence and on the other, they are important to study one of the main objects of M-theory (M2-branes). All these theories have something in common: they can be written in terms of 3-Leibniz algebras. Here we study the structure theory of such algebras, paying special attention to a subclass of them that gives rise to maximal supersymmetry and that was the first to appear in this context: 3-Lie algebras. In chapter 2, we review the structure theory of metric Lie algebras and their unitary representations. In chapter 3, we study metric 3-Leibniz algebras and show, by specialising a construction originally due to Faulkner, that they are in one to one correspondence with pairs of real metric Lie algebras and unitary representations of them. We also show a third characterisation for six extreme...

  13. Fermi surface behavior in the ABJM M2-brane theory

    Science.gov (United States)

    DeWolfe, Oliver; Henriksson, Oscar; Rosen, Christopher

    2015-06-01

    We calculate fermionic Green's functions for states of the three-dimensional Aharony-Bergman-Jafferis-Maldacena M2-brane theory at large N using the gauge-gravity correspondence. We embed extremal black brane solutions in four-dimensional maximally supersymmetric gauged supergravity, obtain the linearized Dirac equations for each spin-1 /2 mode that cannot mix with a gravitino, and solve these equations with infalling boundary conditions to calculate retarded Green's functions. For generic values of the chemical potentials, we find Fermi surfaces with universally non-Fermi liquid behavior, matching the situation for four-dimensional N =4 super-Yang-Mills. Fermi surface singularities appear and disappear discontinuously at the point where all chemical potentials are equal, reminiscent of a quantum critical point. One limit of parameter space has zero entropy at zero temperature, and fermionic fluctuations are perfectly stable inside an energy region around the Fermi surface. An ambiguity in the quantization of the fermions is resolved by supersymmetry.

  14. 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.

  15. Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells.

    Science.gov (United States)

    Lee, Seoung Hoon; Kim, Sangduk; Park, Seung Chul; Kim, Min Ja

    2002-01-01

    Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans. Leptospira sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of Leptospira interrogans serovar lai, a homologue of L. borgpetersenii sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19-28, 2000). Escherichia coli lysate harboring the sphH showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the Leptospira hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of Leptospira SphH was due to the pore formation on mammalian cell membranes.

  16. Granulin exacerbates lupus nephritis via enhancing macrophage M2b polarization.

    Directory of Open Access Journals (Sweden)

    Xi Chen

    Full Text Available BACKGROUND AND AIMS: Lupus nephritis (LN, with considerable morbidity and mortality, is one of the most severe manifestations of systemic lupus erythematosus (SLE. Yet, the pathogenic mechanisms of LN have not been clearly elucidated, and efficient therapies are still in great need. Granulin (GRN, a multifunctional protein linked to inflammatory diseases, has recently been reported to correlate with the disease activity of autoimmune diseases. However, the role of GRN in the pathogenic process of LN still remains obscure. In this study, we explored its potential role and underlying mechanism in the pathogenesis of LN. METHODOLOGY/PRINCIPAL FINDINGS: We found that serum GRN levels were significantly up-regulated and were positively correlated with the severity of LN. Overexpression of GRN in vivo by transgenic injection remarkably exacerbated LN, whereas down-regulation of GRN with shRNA ameliorated LN, firmly demonstrating the critical role of GRN in the pathogenesis of LN. Notably, macrophage phenotype analysis revealed that overexpression of GRN could enhance macrophage polarization to M2b, a key mediator of the initiation and progression of LN. On the contrary, down-regulation of GRN resulted in impaired M2b differentiation, thus ameliorating LN. Moreover, we found that MAPK signals were necessary for the effect of GRN on macrophage M2b polarization. CONCLUSION/SIGNIFICANCE: We first demonstrated that GRN could aggravate lupus nephritis (LN via promoting macrophage M2b polarization, which might provide insights into the pathogenesis of LN as well as potential therapeutic strategies against LN.

  17. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    Directory of Open Access Journals (Sweden)

    Adam L Vanarsdall

    2016-04-01

    Full Text Available Human cytomegalovirus (HCMV is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1 it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  18. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    Science.gov (United States)

    Vanarsdall, Adam L; Howard, Paul W; Wisner, Todd W; Johnson, David C

    2016-04-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  19. Influence of polysaccharides on the rate of coalescence in oil-in-water emulsions formed with highly hydrolyzed whey proteins.

    Science.gov (United States)

    Ye, Aiqian; Hemar, Yacine; Singh, Harjinder

    2004-08-25

    The objective of this study was to examine the effects of added xanthan gum, guar gum, or kappa-carrageenan on the formation and properties of emulsions (4 wt % corn oil) formed with an extensively hydrolyzed commercial whey protein (WPH) product under a range of conditions. The rate of coalescence was calculated on the basis of the changes in the droplet size of emulsions during storage of the emulsions at 20 degrees C. Compared with the emulsion made without the addition of polysaccharides, the rate of creaming and coalescence in emulsions containing xanthan gum, guar gum, or kappa-carrageenan was markedly enhanced with increasing concentration of polysaccharides during storage for up to 7 days. At a given concentration, the rate of coalescence was highest in the emulsions containing guar gum, whereas it was lowest in the emulsions containing kappa-carrageenan. All emulsions containing xanthan gum, guar gum, or kappa-carrageenan showed flocculation of oil droplets by a depletion mechanism. This flocculation was considered to enhance the coalescence of oil droplets. The different rates of coalescence could be explained on the basis of the strength of the depletion potential, which was dependent on the molecular weight and the radius of gyration of the polysaccharides.

  20. A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

    Directory of Open Access Journals (Sweden)

    Lindsay D. Smith

    2014-10-01

    Full Text Available The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.

  1. The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ.

    Science.gov (United States)

    Skoog, Karl; Daley, Daniel O

    2012-02-21

    ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.

  2. Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.

    Science.gov (United States)

    Kim, Kye-Won; Smith, Clyde A; Daily, Michael D; Cort, John R; Davin, Laurence B; Lewis, Norman G

    2015-01-16

    Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (-)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.

  3. Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 Å Resolution with Three Isolated Active Sites*

    Science.gov (United States)

    Kim, Kye-Won; Smith, Clyde A.; Daily, Michael D.; Cort, John R.; Davin, Laurence B.; Lewis, Norman G.

    2015-01-01

    Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (−)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition. PMID:25411250

  4. A simple and fast method to study the hydrodynamic size difference of protein disulfide isomerase in oxidized and reduced form using gold nanoparticles and dynamic light scattering.

    Science.gov (United States)

    Zheng, Tianyu; Cherubin, Patrick; Cilenti, Lucia; Teter, Ken; Huo, Qun

    2016-02-07

    The hydrodynamic dimension of a protein is a reflection of both its molecular weight and its tertiary structures. Studying the hydrodynamic dimensions of proteins in solutions can help elucidate the structural properties of proteins. Here we report a simple and fast method to measure the hydrodyamic size of a relatively small protein, protein disulfide isomerase (PDI), using gold nanoparticle probes combined with dynamic light scattering. Proteins can readily adsorb to citrate-capped gold nanoparticles to form a protein corona. By measuring the average diameter of the gold nanoparticles before and after protein corona formation, the hydrodynamic diameter of the protein can be deduced from the net particle size increase of the assay solution. This study found that when the disulfide bonds in PDI are reduced to thiols, the reduced PDI exhibits a smaller hydrodynamic diameter than the oxided PDI. This finding is in good agreement with the X-ray diffraction analysis of PDI in single crystals. In comparison with other techniques that are used for protein hydrodynamic size analysis, the current method is easy to use, requires a trace amount of protein samples, with results obtained in minutes instead of hours.

  5. Joint TAS and Power Allocation for SDF Relaying M2M Cooperative Networks

    Directory of Open Access Journals (Sweden)

    Lingwei Xu

    2016-01-01

    Full Text Available The outage probability (OP performance of multiple-relay-based selective decode-and-forward (SDF relaying mobile-to-mobile (M2M networks with transmit antenna selection (TAS over N-Nakagami fading channels is investigated. The exact closed-form expressions for OP of the optimal and suboptimal TAS schemes are derived. The power allocation problem is formulated for performance optimization. Then, the OP performance under different conditions is evaluated through numerical simulations to verify the analysis. The simulation results showed that optimal TAS scheme has a better OP performance than suboptimal TAS scheme. Further, the power allocation parameter has an important influence on the OP performance.

  6. 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.

  7. Structural dissection of human translation elongation factor 1Bγ (eEF1Bγ: expression of full-length protein and its truncated forms

    Directory of Open Access Journals (Sweden)

    Trosiuk T. V.

    2014-03-01

    Full Text Available Aim. To gain more insights into properties of the human translation elongation factor eEF1Bγ and its interaction with partners we intended to produce the full-length protein and its truncated forms. Methods. cDNAs encoding truncated forms of eEF1Bγ were generated by PCR amplification with respective primers and cloned into vectors providing polyhistidine, glutathione S-transferase or maltose binding protein tags. The recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography. An aggregation state of the proteins was analyzed by analytical gel filtration. Results. The expression, purification and storage conditions for the full-length recombinant His-eEF1Bγ were optimized. Several truncated forms of eEF1Bγ were also expressed and purified to homogeneity. Two short variants of C-terminal domain comprising amino acids 263–437 or 228–437 were obtained in monomeric state. Two short variants of N-terminal domain comprising amino acids 1–33 or 1–230, fused with glutathione S-transferase, were obtained and estimated to be dimers by gel filtration. The mutants of N-terminal domain comprising amino acids 1–93 or 1–165, fused with maltose binding protein, were obtained as soluble high molecular weight aggregates only. Conclusions. The purified recombinant His-eEF1Bγ and several truncated forms of the protein were obtained and characterized. These protein variants will be used for further studies on the protein-protein interaction.

  8. Telmisartan prevention of LPS-induced microglia activation involves M2 microglia polarization via CaMKKβ-dependent AMPK activation.

    Science.gov (United States)

    Xu, Yuan; Xu, Yazhou; Wang, Yurong; Wang, Yunjie; He, Ling; Jiang, Zhenzhou; Huang, Zhangjian; Liao, Hong; Li, Jia; Saavedra, Juan M; Zhang, Luyong; Pang, Tao

    2015-11-01

    Brain inflammation plays an important role in the pathophysiology of many psychiatric and neurological diseases. During brain inflammation, microglia cells are activated, producing neurotoxic molecules and neurotrophic factors depending on their pro-inflammatory M1 and anti-inflammatory M2 phenotypes. It has been demonstrated that Angiotensin II type 1 receptor blockers (ARBs) ameliorate brain inflammation and reduce M1 microglia activation. The ARB telmisartan suppresses glutamate-induced upregulation of inflammatory genes in cultured primary neurons. We wished to clarify whether telmisartan, in addition, prevents microglia activation through polarization to an anti-inflammatory M2 phenotype. We found that telmisartan promoted M2 polarization and reduced M1 polarization in LPS-stimulated BV2 and primary microglia cells, effects partially dependent on PPARγ activation. The promoting effects of telmisartan on M2 polarization, were attenuated by an AMP-activated protein kinase (AMPK) inhibitor or AMPK knockdown, indicating that AMPK activation participates on telmisartan effects. Moreover, in LPS-stimulated BV2 cells, telmisartan enhancement of M2 gene expression was prevented by the inhibitor STO-609 and siRNA of calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Furthermore, telmisartan enhanced brain AMPK activation and M2 gene expression in a mouse model of LPS-induced neuroinflammation. In addition, telmisartan reduced the LPS-induced sickness behavior in this in vivo model, and this effect was prevented by prior administration of an AMPK inhibitor. Our results indicate that telmisartan can be considered as a novel AMPK activator, suppressing microglia activation by promoting M2 polarization. Telmisartan may provide a novel, safe therapeutic approach to treat brain disorders associated with enhanced inflammation.

  9. 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.

  10. [NEW INFORMATION ABOUT THE STRUCTURES FORMED BY FtsZ PROTEIN IN ESCHERICHIA COLI CELLS DURING DIVISION PROCESS OBTAINED BY SINGLE-MOLECULE LOCALIZATION MICROSCOPY].

    Science.gov (United States)

    Vedyaykin, A D; Vishnyakov, I E; Polinovskaya, V S; Artamonova, I T; Khodorkovskii, M A; Sabantsev, A V

    2015-01-01

    FtsZ--a bacterial tubulin homolog--is one of the key bacterial division proteins, forming a contractile Z-ring at the midcell of dividing bacteria. In this work immunofluorescent labeling was used in conjunction with single-molecule localization microscopy (SMLM) to visualize native structures formed by FtsZ protein in Escherichia coli cells. This approach allowed the reorganization of FtsZ structures during cytokinesis to be visualized step-by-step. New data was obtained that support the hypothesis that the Z-ring is a spiral structure that constricts during division, assisting the formation of the septum between daughter cells.

  11. Expression, purification, crystallization and preliminary X-ray characterization of two crystal forms of stationary-phase survival E protein from Campylobacter jejuni

    Energy Technology Data Exchange (ETDEWEB)

    Gonçalves, A. M. D.; Rêgo, A. T. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Thomaz, M. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2748-901 Oeiras (Portugal); Enguita, F. J., E-mail: fenguita@fm.ul.pt [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal); Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa (Portugal); Carrondo, M. A. [Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras (Portugal)

    2008-03-01

    Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. The first form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 Å. The second form belongs to space group C2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 Å, and contains a dimer in the asymmetric unit. Diffraction data have been collected from these crystal forms to 2.5 and 2.95 Å resolution, respectively.

  12. Coexistence of two forms of disease-associated prion protein in extracerebral tissues of cattle infected with H-type bovine spongiform encephalopathy.

    Science.gov (United States)

    Okada, Hiroyuki; Miyazawa, Kohtaro; Masujin, Kentaro; Yokoyama, Takashi

    2016-08-01

    H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in aged cattle. H-BSE is characterized by the presence of two proteinase K-resistant forms of disease-associated prion protein (PrP(Sc)), identified as PrP(Sc) #1 and PrP(Sc) #2, in the brain. To investigate the coexistence of different PrP(Sc) forms in the extracerebral tissues of cattle experimentally infected with H-BSE, immunohistochemical and molecular analyses were performed by using N-terminal-, core-region- and C-terminal-specific anti-prion protein antibodies. Our results demonstrated that two distinct forms of PrP(Sc) coexisted in the various extracerebral tissues.

  13. 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

    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...... 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...

  14. Experimental and numerical studies of the chromatofocusing of dilute proteins using retained pH gradients formed on a strong-base anion-exchange column.

    Science.gov (United States)

    Strong, J C; Frey, D D

    1997-05-09

    The separation of dilute protein mixtures was achieved using simple monovalent buffering species to form retained, internally produced pH gradients on a strong-basic anion-exchange column. Highly focused proteins bands localized on stepwise pH transitions were produced experimentally under trace and volume overloaded feed conditions. Numerical simulations were performed that accurately predict the pH profile and protein band shapes in the column effluent. Experimental results were combined with numerical investigations to explore strategies for designing efficient preparative-scale chromatofocusing systems using simple, inexpensive buffers and adsorbents.

  15. Reseach on Business Model of M2M Virtual Opertion%M2M虚拟运营的商业模式研究

    Institute of Scientific and Technical Information of China (English)

    景士颖; 乔新春

    2014-01-01

    M2M市场在未来几年拥有较大的增长空间,随着移动转售业务牌照的发放,M2M将成为虚拟运营商可以切入的具备潜力的市场领域之一。探讨M2M虚拟运营的商业模式,分析国际开展M2M虚拟运营的案例,并给出国内开展M2M虚拟运营的建议。%M2M has more market space for growth in the coming years, with the issuance of mobile resale service license, M2M wil become a potential market area where MVNOs can cut into. This article discusses the business model of M2M virtual operation, analyzes the international M2M MVNO business cases, and gives advices to MNOs and MVNOs on how to provide better M2M services in China.

  16. Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors.

    Science.gov (United States)

    Daeffler, L; Schmidlin, F; Gies, J P; Landry, Y

    1999-03-01

    1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*pirenzepine was R*>R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.

  17. Pharmacological effects of mitraphylline from Uncaria tomentosa in primary human monocytes: Skew toward M2 macrophages.

    Science.gov (United States)

    Montserrat-de la Paz, S; de la Puerta, R; Fernandez-Arche, A; Quilez, A M; Muriana, F J G; Garcia-Gimenez, M D; Bermudez, B

    2015-07-21

    Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC. (Rubiaceae) is a Peruvian thorny liana, commonly known as "cat׳s claw", and traditionally used in folk medicine to deal with several inflammatory diseases. Mitraphylline (MTP) is the most abundant pentacyclic oxindolic alkaloid (POA) from U. Tomentosa and has been reported to modify the inflammatory response. Herein, we have sought to identify the mechanisms underlying this modulatory effect of MTP on primary human monocytes and its ability to regulate differentiation processes on human primary monocyte and monocyte-derived macrophages. In vitro studies with human primary monocytes and monocyte-derived macrophages were performed. Monocytes and M0 macrophages were exposed to MTP (25μM) and LPS (100ng/mL). M0 macrophages were polarized to M1 and M2 phenotypes in the absence or presence of MTP. The activation state of monocytes/macrophages was assessed by flow cytometry, gene expression and protein analysis of different specific markers. In human primary monocytes, the incubation of MTP for 24h reduced the number of classical (CD14(++)CD16(-)) and intermediate (CD14(++)CD16(+)) subsets when compared to untreated or LPS-treated cells. MTP also reduced the chemotactic capacity of human primary monocytes. In addition, MTP promoted the polarization of M0 macrophages toward an anti-inflammatory M2 phenotype, the abrogation of the release of pro-inflammatory cytokines such as TNFα, IL-6 or IL-1β, as well as the restoration of markers for M2 macrophages in LPS-treated M1 macrophages. Our results suggest that MTP may be a key modulator for regulating the plasticity of monocytes/macrophages and the attenuation of the inflammatory response. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Siti H Sheikh Abdul Kadir

    Full Text Available BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC, which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM. Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart. METHODS AND RESULTS: Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2 receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2 receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters. CONCLUSION: We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2 receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

  19. Rebuilding a macromolecular membrane complex at the atomic scale: case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2.

    Science.gov (United States)

    Sapay, Nicolas; Estrada-Mondragon, Argel; Moreau, Christophe; Vivaudou, Michel; Crouzy, Serge

    2014-09-01

    Ion channel-coupled receptors (ICCR) are artificial proteins built from a G protein-coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high-throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2-Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2-Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2-Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ-opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2-Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N-terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2-Kir6.2 construct.

  20. Conformational characterization of synapse-associated protein 97 by nuclear magnetic resonance and small-angle X-ray scattering shows compact and elongated forms.

    Science.gov (United States)

    Tully, Mark D; Grossmann, J Günter; Phelan, Marie; Pandelaneni, Sravan; Leyland, Mark; Lian, Lu-Yun

    2012-01-31

    Synapse-associated protein 97 (SAP97) is a membrane-associated guanylate kinase protein that interacts with other proteins such as ion channels, subunits of glutamate receptors, and other cytoskeletal proteins and molecular scaffolds. The molecular diversity of SAP97 results from alternative splicing at the N-terminus, and in the U1 and U5 regions. There are two main N-terminal isoforms: the β-isoform has an L27 domain, whereas in the α-isoform, this is replaced by a palmitoylation motif. We have used multiangle light scattering, nuclear magnetic resonance, and small-angle X-ray scattering studies to characterize the conformation of a truncated form of the β-isoform, hence mimicking the α-isoform. This paper provides a comprehensive view of the small-angle X-ray scattering data, and the resulting data show that the scattering data are consistent with the presence of an ensemble of forms in dynamic equilibrium, with two prominent populations of compact and extended forms, with R(g) values of 38 ± 7 Å (52%) and 70 ± 10 Å (37%), respectively. The data show that without the L27 domain, the conformation of SAP97 is biased toward the compact form. We propose a hypothesis in which the overall conformation of SAP97 is determined by the nature of the N-terminus, which may, in turn, influence the specific role of a particular splice variant.

  1. 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.

  2. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  3. 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

    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 (S1P1–5) 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 S1P1. In contrast, M1-polarized macrophages significantly downregulated S1P4. 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.

  4. 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 OD420 (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 OD420 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.V. All

  5. Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.

    Science.gov (United States)

    Bornemann, D; Miller, E; Simon, J

    1998-10-01

    The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.

  6. 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

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    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 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-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels 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 in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  7. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    Science.gov (United States)

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  8. The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix.

    Science.gov (United States)

    Vázquez, M I; Rivas, G; Cregut, D; Serrano, L; Esteban, M

    1998-12-01

    The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and

  9. Haemophilus ducreyi-induced interleukin-10 promotes a mixed M1 and M2 activation program in human macrophages.

    Science.gov (United States)

    Li, Wei; Katz, Barry P; Spinola, Stanley M

    2012-12-01

    During microbial infection, macrophages are polarized to classically activated (M1) or alternatively activated (M2) cells in response to microbial components and host immune mediators. Proper polarization of macrophages is critical for bacterial clearance. To study the role of macrophage polarization during Haemophilus ducreyi infection, we analyzed a panel of macrophage surface markers in skin biopsy specimens of pustules obtained from experimentally infected volunteers. Lesional macrophages expressed markers characteristic of both M1 and M2 polarization. Monocyte-derived macrophages (MDM) also expressed a mixed M1 and M2 profile of surface markers and cytokines/chemokines upon infection with H. ducreyi in vitro. Endogenous interleukin 10 (IL-10) produced by infected MDM downregulated and enhanced expression of several M1 and M2 markers, respectively. Bacterial uptake, mediated mainly by class A scavenger receptors, and activation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways were required for H. ducreyi-induced IL-10 production in MDM. Compared to M1 cells, IL-10-polarized M2 cells displayed enhanced phagocytic activity against H. ducreyi and similar bacterial killing. Thus, IL-10-modulated macrophage polarization may contribute to H. ducreyi clearance during human infection.

  10. High-resolution structures of the D-alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo- and holo-forms.

    Science.gov (United States)

    Zimmermann, Stephan; Pfennig, Sabrina; Neumann, Piotr; Yonus, Huma; Weininger, Ulrich; Kovermann, Michael; Balbach, Jochen; Stubbs, Milton T

    2015-08-19

    D-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The D-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales.

  11. GRAS Proteins Form a DNA Binding Complex to Induce Gene Expression during Nodulation Signaling in Medicago truncatula

    National Research Council Canada - National Science Library

    Sibylle Hirsch; Jiyoung Kim; Alfonso Muñoz; Anne B. Heckmann; J. Allan Downie; Giles E. D. Oldroyd

    2009-01-01

    .... Perception of the calcium oscillations is a function of a calcium-and calmodulin-dependent protein kinase, and this activates nodulation gene expression via two GRAS domain transcriptional regulators...

  12. In vivo reduction-oxidation state of protein disulfide isomerase: the two active sites independently occur in the reduced and oxidized forms

    DEFF Research Database (Denmark)

    Appenzeller-Herzog, Christian; Ellgaard, Lars

    2008-01-01

    by the redox state of active-site cysteines found in a Cys-Xaa-Xaa-Cys motif. Progress in understanding redox regulation of the mammalian enzymes is currently hampered by the lack of reliable methods to determine quantitatively their redox state in living cells. We developed such a method based......Thiol-disulfide oxidoreductases of the human protein disulfide isomerase (PDI) family promote protein folding in the endoplasmic reticulum (ER), while also assisting the retrotranslocation of toxins and misfolded ER proteins to the cytosol. The redox activity of PDI-like proteins is determined...... on the alkylation of cysteines by methoxy polyethylene glycol 5000 maleimide. With this method, we showed for the first time that in vivo PDI is present in two semi-oxidized forms in which either the first active site (in the a domain) or the second active site (in the a' domain) is oxidized. We report a steady...

  13. Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel.

    Science.gov (United States)

    Geng, Shuhui; White, Shane N; Paine, Michael L; Snead, Malcolm L

    2015-08-21

    Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.

  14. Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function.

    Science.gov (United States)

    Tan, William; Schauder, Curtis; Naryshkina, Tatyana; Minakhina, Svetlana; Steward, Ruth

    2016-02-15

    Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. In this study, we identified FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. We show that Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, we demonstrate that Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. We propose that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus.

  15. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype.

    Science.gov (United States)

    Ichiyama, Susumu; Oka, Yoshiaki; Haga, Kazuko; Kojima, Shuichi; Tateishi, Yukihiro; Shirakawa, Masahiro; Haga, Tatsuya

    2006-01-09

    We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

  16. Exact relations between M2-brane theories with and without Orientifolds

    CERN Document Server

    Honda, Masazumi

    2015-01-01

    We study partition functions of low-energy effective theories of M2-branes, whose type IIB brane constructions include orientifolds. We mainly focus on circular quiver superconformal Chern-Simons theory on S^3, whose gauge group is O(2N+1)x USp(2N)x ... x O(2N+1)x USp(2N). This theory is a natural generalization of N=5 ABJM theory with the gauge group O(2N+1)_{2k}x USp(2N)_{-k}. We find that the partition function of this type of theory has a simple relation to the one of the M2-brane theories without the orientifolds, whose gauge group is U(N)x ... x U(N). By using this relation, we determine an exact form of the grand partition function of the O(2N+1)_2 x USp(2N)_{-1} ABJM theory, where its supersymmetry is expected to enhance to N=6. As another interesting application, we discuss that our result gives a natural physical interpretation of a relation between grand partition functions of the U(N+1)_4 x U(N)_{-4} ABJ theory and U(N)_2 x U(N)_{-2} ABJM theory, recently conjectured by Grassi-Hatsuda-Marino. We a...

  17. 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

  18. 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.

  19. Supplemented vaccination with tandem repeat M2e virus-like particles enhances protection against homologous and heterologous HPAI H5 viruses in chickens.

    Science.gov (United States)

    Song, Byung-Min; Kang, Hyun-Mi; Lee, Eun-Kyoung; Jung, Suk Chan; Kim, Min-Chul; Lee, Yu-Na; Kang, Sang-Moo; Lee, Youn-Jeong

    2016-01-27

    Highly pathogenic avian influenza (HPAI) H5 viruses derived from A/Goose/Guangdong/1/96 have been continuously circulating globally, severely affecting the public health and poultry industries. The matrix 2 protein ectodomain (M2e) is considered a promising candidate for a universal cross-protective influenza vaccine that provides more effective control over HPAI H5 viruses harboring variant hemagglutinin (HA)-antigens. Here, we evaluated the protective efficacy of a tandem repeat construct of heterologous M2e presented on virus-like particles (M2e5x VLPs) either alone or as a supplement against HPAI H5 viruses in a chicken model. Chickens immunized with M2e5x VLPs alone induced M2e-specific antibodies but were not protected against HPAI H5. The homo- and cross-protective efficacy of M2e5x VLP-supplemented vaccination of chickens was also examined. Importantly, supplementation with M2e5x VLPs induced significantly higher levels of antibodies specific for M2e and different viruses as well as provided improved protection against homologous and heterologous HPAI H5 viruses. Considering the limited efficacy of inactivated vaccines, supplement vaccination with M2e5x VLPs may be an effective measure for preventing outbreaks of HPAI viruses that have the ability to constantly change their antigenic properties in poultry.

  20. Stable free-radical forms of plasma proteins or simpler related structures which induce brain excitatory effects.

    Science.gov (United States)

    Polis, B D; Wyeth, J; Goldstein, L; Graedon, J

    1969-10-01

    Stable free radicals have been prepared from purified plasma proteins, pituitary peptides, and simpler related structures like 5-OH tryptophan and melatonin by oxidation with the free-radical nitrosyl disulfonate in alkaline solution under controlled conditions. The presence of tyrosine or trytophan amino acid residues in the protein was found essential for free-radical formation. These red-colored, stable free radicals showed electron spin resonance spectra in aqueous solutions at room temperature and maintained this characteristic for weeks when stored at 5 degrees C. Illumination, by visible light, of the free-radical proteins and peptides separated from excess nitrosyl disulfonate by salt fractionation or chromatography enhanced the free-radical concentration in the light. The increased signal decayed in the dark. Intravenous administration of the free-radical proteins or peptides into rabbits equipped with chronic cranial electrodes and sedated with a small dose of pentobarbital caused a sudden EEG arousal accompanied by behavioral changes indicative of brain excitation. Illumination of the free-radical compounds prior to administration enhanced the effects. Untreated control proteins or peptides had no effects. The observations are interpreted to suggest the involvement of free-radical structures in the transfer of energy in nervous tissue.

  1. A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

    Science.gov (United States)

    Mayer, T; Meyer, M; Janning, A; Schiedel, A C; Barnekow, A

    1999-03-25

    The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

  2. A ssDNA Aptamer That Blocks the Function of the Anti-FLAG M2 Antibody

    Directory of Open Access Journals (Sweden)

    Amanda S. Lakamp

    2011-01-01

    Full Text Available Using SELEX (systematic evolution of ligands by exponential enrichment, we serendipitously discovered a ssDNA aptamer that binds selectively to the anti-FLAG M2 antibody. The aptamer consisted of two motifs (CCTTA and TGTCTWCC separated by 2-3 bases, and the elimination of one or the other motif abrogated binding. The DNA aptamer and FLAG peptide competed for binding to the antigen-binding pocket of the M2 antibody. In addition, the aptamer eluted FLAG-tagged proteins from the antibody, suggesting a commercial application in protein purification. These findings demonstrate the feasibility of using SELEX to develop ssDNA aptamers that block the function of a specific antibody, a capability that could lead to the development of novel therapeutic modalities for patients with systemic lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases.

  3. 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...... means that form serves both as the connective value and as the concept for reflection. In other words, form is observed as form, not anything else. The didactical challenge of teaching form as form is accentuated by students’ everyday-based pre-orientation towards function at the expense of form....... In general, students enter design education as far more skilled observers with regards to function than form. They are, in other words, predisposed to observe objects asking ‘what is?’, rather than ‘how is?’. This habit has not only cognitive implications. It is closely intertwined with a rudimentary...

  4. 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...... means that form serves both as the connective value and as the concept for reflection. In other words, form is observed as form, not anything else. The didactical challenge of teaching form as form is accentuated by students’ everyday-based pre-orientation towards function at the expense of form...... vocabulary of form. Even in cases in which teaching uses terms and phrases from everyday life (for instance, ‘intersection’), the meaning of the word cannot necessarily be transmitted directly from an ordinary vocabulary into a design context. And it is clearly a common issue for the contributions...

  5. The irre cell recognition module (IRM) protein Kirre is required to form the reciprocal synaptic network of L4 neurons in the Drosophila lamina.

    Science.gov (United States)

    Lüthy, Kevin; Ahrens, Birgit; Rawal, Shilpa; Lu, Zhiyuan; Tarnogorska, Dorota; Meinertzhagen, Ian A; Fischbach, Karl-Friedrich

    2014-01-01

    Each neuropil module, or cartridge, in the fly's lamina has a fixed complement of cells. Of five types of monopolar cell interneurons, only L4 has collaterals that invade neighboring cartridges. In the proximal lamina, these collaterals form reciprocal synapses with both the L2 of their own cartridge and the L4 collateral branches from two other neighboring cartridges. During synaptogenesis, L4 collaterals strongly express the cell adhesion protein Kirre, a member of the irre cell recognition module (IRM) group of proteins ( Fischbach et al., 2009 , J Neurogenet, 23, 48-67). The authors show by mutant analysis and gene knockdown techniques that L4 neurons develop their lamina collaterals in the absence of this cell adhesion protein. Using electron microscopy (EM), the authors demonstrate, however, that without Kirre protein these L4 collaterals selectively form fewer synapses. The collaterals of L4 neurons of various genotypes reconstructed from serial-section EM revealed that the number of postsynaptic sites was dramatically reduced in the absence of Kirre, almost eliminating any synaptic input to L4 neurons. A significant reduction of presynaptic sites was also detected in kirre(0) mutants and gene knockdown flies using RNA interference. L4 neuron reciprocal synapses are thus almost eliminated. A presynaptic marker, Brp-short(GFP) confirmed these data using confocal microscopy. This study reveals that removing Kirre protein specifically disrupts the functional L4 synaptic network in the Drosophila lamina.

  6. 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.

  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. Dy$^{3+}$-activated M$_2$SiO$_4$ (M $=$ Ba, Mg, Sr)-type phosphors

    Indian Academy of Sciences (India)

    ESRA ÖZTÜRK; ERKUL KARACAOGLU

    2017-02-01

    The alkaline orthosilicates of M$_2$SiO$_4$ (M $=$ Ba, Mg, Sr) activated with Dy$^{3+}$ and co-doped with Ho$^{3+}$ are prepared through conventional solid-state method, i.e., mixing and grinding of solid form precursors followedby high-temperature heat treatments of several hours in furnaces, generally under open atmosphere and investigated by X-ray diffraction (XRD) to get phase properties and photoluminescence (PL) analysis to get luminescenceproperties. The thermal behaviours of well-mixed samples were determined by differential thermal analysis (DTA)/thermogravimetry (TG). The PL spectra show that the 478 and 572nm maximum emission bands are attributed, respectively, to ${}^{4}$F$_{9/2}$ $\\to$ ${}^{6}$H$_{15/2}$ and ${}^{4}$F$_{9/2}$ $\\to$ ${}^{6}$H$_{13/2}$ transitions of Dy$^{3+}$ ions.

  9. Characterization of the various forms of the Reelin protein in the cerebrospinal fluid of normal subjects and in neurological diseases.

    Science.gov (United States)

    Ignatova, Nina; Sindic, Christian J M; Goffinet, André M

    2004-03-01

    Reelin is a large extracellular glycoprotein that is defective in reeler mutant mice and plays a well-established role during brain development in human as well as rodents. In the adult brain, Reelin is expressed in a subset of GABAergic interneurons. Its role in disease states is not clearly defined, although it is implicated in autism and psychoses such as schizophrenia. In this report, we show that Reelin immunoreactive proteins can be detected in the human cerebrospinal fluid (CSF) with monoclonal antibodies directed against the N- and C-terminal regions of the protein. In CSF, Reelin is present as different products due to processing at two main sites; preservation at -20 degrees C increases processing further. CSF Reelin originates from the brain tissue and not from plasma. The protein was detected in comparable concentrations in children and adults, and the signal varied largely from subject to subject with no obvious correlation with age or neurological disease state.

  10. Selective Protein Extraction from Chlorobium tepidum Chlorosomes Using Detergents. Evidence That CsmA Forms Multimers and Binds Bacteriochlorophyll a

    DEFF Research Database (Denmark)

    Bryant, Donald A; Vassilieva, Elena V; Frigaard, Niels-Ulrik

    2002-01-01

    and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters....... When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes....... Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape...

  11. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    Science.gov (United States)

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 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.

  13. Selective protein extraction from Chlorobium tepidum chlorosomes using detergents. Evidence that CsmA forms multimers and binds bacteriochlorophyll a.

    Science.gov (United States)

    Bryant, Donald A; Vassilieva, Elena V; Frigaard, Niels-Ulrik; Li, Hui

    2002-12-03

    Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.

  14. 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.

  15. Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice.

    Directory of Open Access Journals (Sweden)

    Amaya I Wolf

    Full Text Available BACKGROUND: The extracellular domain of the influenza A virus protein matrix protein 2 (M2e is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab secreting cells (ASCs and Ab isotypes, and tested the protective efficacy in various mouse strains. METHODOLOGY/PRINCIPAL FINDINGS: Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN and cholera toxin (CT elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c. and intranasal (i.n. delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL. Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs. CONCLUSION/SIGNIFICANCE: Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human

  16. 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 Cx31.

  17. 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 Cx31.

  18. M2M在3GPP SA2的研究进展%The Progress of M2M in 3GPP SA2

    Institute of Scientific and Technical Information of China (English)

    杜加懂

    2011-01-01

    With the rapid development of Internet of things,almost all communication standardization organizations put their focus on the standards of M2M.3GPP as the main mobile communication SDO is working hard on research and standards on M2M.Based on the brief introduction of the 3GPP working groups on M2M,this paper mainly introduces the 3GPP SA2 research progress and standardization on M2M.%随着物联网的快速发展,M2M成为各个标准化组织研究和标准制定的工作重点。3GPP作为移动通信技术的主要研究和标准制定者,对M2M的相关研究和标准制定也在加紧进行。本文在介绍3GPP各个工作组的工作情况的基础上,重点介绍了M2M在3GPP在SA2的研究和标准化进展情况。

  19. File list: ALL.Myo.50.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.50.AllAg.LHCN-M2 hg19 All antigens Muscle LHCN-M2 SRX201296,SRX201297,SRX19...3596,SRX193611,SRX201281,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.50.AllAg.LHCN-M2.bed ...

  20. File list: DNS.Myo.10.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Myo.10.AllAg.LHCN-M2 hg19 DNase-seq Muscle LHCN-M2 SRX201296,SRX201297,SRX19359...6,SRX201281,SRX193611,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Myo.10.AllAg.LHCN-M2.bed ...

  1. File list: ALL.Myo.05.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.05.AllAg.LHCN-M2 hg19 All antigens Muscle LHCN-M2 SRX201296,SRX201297,SRX19...3596,SRX201281,SRX193611,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.05.AllAg.LHCN-M2.bed ...

  2. File list: DNS.Myo.50.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Myo.50.AllAg.LHCN-M2 hg19 DNase-seq Muscle LHCN-M2 SRX201296,SRX201297,SRX19359...6,SRX193611,SRX201281,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Myo.50.AllAg.LHCN-M2.bed ...

  3. File list: ALL.Myo.10.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.10.AllAg.LHCN-M2 hg19 All antigens Muscle LHCN-M2 SRX201296,SRX201297,SRX19...3596,SRX201281,SRX193611,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.10.AllAg.LHCN-M2.bed ...

  4. File list: ALL.Myo.20.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.20.AllAg.LHCN-M2 hg19 All antigens Muscle LHCN-M2 SRX201296,SRX201297,SRX19...3596,SRX201281,SRX193611,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Myo.20.AllAg.LHCN-M2.bed ...

  5. File list: DNS.Myo.20.AllAg.LHCN-M2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Myo.20.AllAg.LHCN-M2 hg19 DNase-seq Muscle LHCN-M2 SRX201296,SRX201297,SRX19359...6,SRX201281,SRX193611,SRX201282 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Myo.20.AllAg.LHCN-M2.bed ...

  6. An assessment of the use of native and denatured forms of okra seed proteins as coagulants in drinking water treatment.

    Science.gov (United States)

    Jones, Alfred Ndahi; Bridgeman, John

    2016-10-01

    The effects of temperature, storage time and water pH on the coagulation performance of okra seed protein in water treatment were assessed. In a jar test experiment, okra salt extract achieved a notable improvement in treatment efficiency with storage time and showed good performance in quality after thermal treatment at 60, 97 and 140 °C temperatures for 6, 4 and 2 hours, respectively. The performance improvement of more than 8% is considered to be due to the denaturation and subsequent removal of coagulation-hindering proteins in okra seed. Furthermore, the results of a sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis show two distinctive bands of protein responsible for the coagulation process after denaturation. It was further shown that at optimal coagulant dose, the pH of the treated water remained unaffected as a result of the protein's buffering capability during coagulation. Therefore, denatured okra seed exhibited improved performance compared to the native crude extract and offers clear benefits as a water treatment coagulant.

  7. M1 and M2 Functional Imprinting of Primary Microglia: Role of P2X7 Activation and miR-125b

    Directory of Open Access Journals (Sweden)

    Chiara Parisi

    2016-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a most frequently occurring and severe form of motor neuron disease, causing death within 3–5 years from diagnosis and with a worldwide incidence of about 2 per 100,000 person-years. Mutations in over twenty genes associated with familial forms of ALS have provided insights into the mechanisms leading to motor neuron death. Moreover, mutations in two RNA binding proteins, TAR DNA binding protein 43 and fused in sarcoma, have raised the intriguing possibility that perturbations of RNA metabolism, including that of the small endogenous RNA molecules that repress target genes at the posttranscriptional level, that is, microRNAs, may contribute to disease pathogenesis. At present, the mechanisms by which microglia actively participate to both toxic and neuroprotective actions in ALS constitute an important matter of research. Among the pathways involved in ALS-altered microglia responses, in previous works we have uncovered the hyperactivation of P2X7 receptor by extracellular ATP and the overexpression of miR-125b, both leading to uncontrolled toxic M1 reactions. In order to shed further light on the complexity of these processes, in this short review we will describe the M1/M2 functional imprinting of primary microglia and a role played by P2X7 and miR-125b in ALS microglia activation.

  8. PPARγ ligands switched high fat diet-induced macrophage M2b polarization toward M2a thereby improving intestinal Candida elimination.

    Directory of Open Access Journals (Sweden)

    Lise Lefèvre

    Full Text Available Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD-induced obesity and the effect of rosiglitazone (PPARγ ligand or WY14643 (PPARα ligand both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-α(high, IL-10(high, MR, Dectin-1. Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-α(low, IL-10(low, MR(high, Dectin-1(high switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level.

  9. SMAD-PI3K-Akt-mTOR pathway mediates BMP-7 polarization of monocytes into M2 macrophages.

    Directory of Open Access Journals (Sweden)

    Crystal Rocher

    Full Text Available Previously we demonstrated that bone morphogenetic protein-7 (BMP-7 treatment polarizes monocytes into M2 macrophages and increases the expression of anti-inflammatory cytokines. Despite these findings, the mechanisms for the observed BMP-7 induced monocyte polarization into M2 macrophages are completely unknown. In this study, we demonstrate the mechanisms involved in the polarization of monocytes into M2 macrophages. Apoptotic conditioned media (ACM was generated to mimic the stressed conditions, inducing monocyte polarization. Monocytes were treated with ACM along with BMP-7 and/or its inhibitor, follistatin, for 48 hours. Furthermore, an inhibitor of the PI3K pathway, LY-294002, was also studied. Our data show that BMP-7 induces polarization of monocytes into M2 macrophages while significantly increasing the expression of anti-inflammatory markers, arginase-1 and IL-10, and significantly (p<0.05 decreasing the expression of pro-inflammatory markers iNOS, IL-6, TNF-α and MCP-1; (p<0.05. Moreover, addition of the PI3K inhibitor, LY-294002, significantly (p<0.05 decreases upregulation of IL-10 and arginase-1, suggesting involvement of the PI3K pathway in M2 macrophage polarization. Next, following BMP-7 treatment, a significant (p<0.05 increase in p-SMAD1/5/8 and p-PI3K expression resulting in downstream activation of p-Akt and p-mTOR was observed. Furthermore, expression of p-PTEN, an inhibitor of the PI3K pathway, was significantly (p<0.05 increased in the ACM group. However, BMP-7 treatment inhibited its expression, suggesting involvement of the PI3K-Akt-mTOR pathway. In conclusion, we demonstrate that BMP-7 polarizes monocytes into M2 macrophages and enhances anti-inflammatory cytokine expression which is mediated by the activated SMAD-PI3K-Akt-mTOR pathway.

  10. Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group.

    Science.gov (United States)

    Kreuze, J F; Karyeija, R F; Gibson, R W; Valkonen, J P

    2000-01-01

    Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) infects sweet potatoes (Ipomoea batatas) worldwide, but no sequence data on isolates from Africa are available. Coat protein (CP) gene sequences from eight East African isolates from Madagascar and different districts of Uganda (the second biggest sweet potato producer in the world) and two West African isolates from Nigeria and Niger were determined. They were compared by phylogenetic analysis with the previously reported sequences of ten SPFMV isolates from other continents. The East African SPFMV isolates formed a distinct cluster, whereas the other isolates were not clustered according to geographic origin. These data indicate that East African isolates of SPFMV form a genetically unique group.

  11. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  12. Pentraxin-3 Attenuates Renal Damage in Diabetic Nephropathy by Promoting M2 Macrophage Differentiation.

    Science.gov (United States)

    Sun, Huaibin; Tian, Jun; Xian, Wanhua; Xie, Tingting; Yang, Xiangdong

    2015-10-01

    As one of the most important long-term complications of diabetes, diabetic nephropathy (DN) is the major cause of end-stage renal disease and high mortality in diabetic patients. The long pentraxin 3 (Ptx3) is a member of a superfamily of conserved proteins characterized by a cyclic multimeric structure and a conserved C-terminal domain. Several clinical investigations have demonstrated that elevated plasma Ptx3 levels are associated with cardiovascular and chronic kidney diseases (CKD). However, the therapeutic effect of Ptx3 on DN has never been investigated. In our current study, we showed a crucial role for Ptx3 in attenuating renal damage in DN. In our mouse hyperglycemia-induced nephropathy model, Ptx3 treatment showed significantly increased expression of nephrin, acetylated nephrin, and Wilm's tumor-1 protein (WT-1) when compared with control. The number of CD4(+) T cells, CD8(+) T cells, Ly6G(+) neutrophils, and CD11b(+) macrophages were all significantly lower in the Ptx3-treated group than that in the control group in DN. The IL-4 and IL-13 levels in the Ptx3-treated group were markedly higher than that in the control group in DN. Correspondingly, the Ptx3-treated group showed increased numbers of Arg1- or CD206-expressing macrophages compared with the control group. Furthermore, inhibition of Ptx3-treated macrophages abrogated the alleviated renal damage induced by Ptx3 treatment. In conclusion, Ptx3 attenuates renal damage in DN by promoting M2 macrophage differentiation.

  13. The research of neurospecific proteins and lysosomal peptidehydrolases in frontal neocortex during forming conditioned reaction of active avoiding of rats

    Directory of Open Access Journals (Sweden)

    Vyatkin O. K.

    2009-04-01

    Full Text Available Dynamics of the activity of neuronal cell adhesion molecule (NCAM and lysosomal cysteine cathepsins B, L, H was researched in frontal neocortex of rat brain during forming a conditioned reaction of active avoiding. The quantitative estimation of NCAM content in the neocortex membrane fraction was carried on by ELISA in 3, 7, 14 and 21 days after starting animals’ training. The dynamics correlation between the NCAM content increasing and cysteine cathepsins activity was obtained, especially for aminopeptidase cathepsin H during the process of memory engram forming in frontal neocortex of rat brain.

  14. Effect of glycerol content and pH value of film-forming solution on the functional properties of protein-based edible films

    Directory of Open Access Journals (Sweden)

    Nemet Nevena T.

    2010-01-01

    Full Text Available The work is concerned with the effects of glycerol content and pH value of film forming solution on the functional properties of protein-based films. The films were produced of chicken breast proteins, dissolved under either acidic (pH 3 or alkaline (pH 11 conditions, with different concentrations of glycerol (35%, 50% and 65% w/w of protein content. Glycerol content affected significantly mechanical properties, water vapor permeability, color at pH 3 and film solubility (p<0.05. The pH value had significant influence on light transmission, color, transparency and film solubility (p<0.05. Considering the results of mechanical properties and film solubility, the obtained films are in the acceptable range for the use as a packaging material. It was estimated that water vapor permeability, color, light transmission and transparency need to be improved for the application.

  15. The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways.

    Science.gov (United States)

    Dhakal, Bijaya K; Mulvey, Matthew A

    2012-01-19

    Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell functions, but also disable macrophages and suppress inflammatory responses.

  16. Definition and measurement of the beam propagation factor M2 for chromatic laser beams

    Institute of Scientific and Technical Information of China (English)

    Tao Fang; Xin Ye; Jinfu Niu; Jianqiu Xu

    2006-01-01

    The concept of the beam propagation factor M2 is extended for chromatic laser beams. The definition of the beam propagation factor can be generalized with the weighted effective wavelength. Using the new definition of factor M2, the propagation of chromatic beams can be analyzed by the beam propagation factor M2 as same as that of monochromatic beams. A simple method to measure the chromatic beam factor M2 is demonstrated. The chromatic factor M2 is found invariable while the laser beam propagates through the dispersion-free ABCD system.

  17. Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

    Science.gov (United States)

    Philippe, Monique; Léger, Thibaut; Desvaux, Raphaëlle; Walch, Laurence

    2013-05-03

    Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

  18. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly*S⃞

    OpenAIRE

    McKee, Karen K.; Capizzi, Stephanie; Yurchenco, Peter D.

    2009-01-01

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the α4 and α5 subunits are expressed in α2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind...

  19. Interleukin-13 receptors on human prostate carcinoma cell lines represent a novel target for a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin.

    Science.gov (United States)

    Maini, A; Hillman, G; Haas, G P; Wang, C Y; Montecillo, E; Hamzavi, F; Pontes, J E; Leland, P; Pastan, I; Debinski, W; Puri, R K

    1997-09-01

    We have discovered a new cell surface protein in the form of interleukin-13 receptor on several solid tumor cells, including human renal cell carcinoma cells (Obiri et al., 1995; Debinski et al., 1995). This study reports that human prostate cancer cell lines also express high affinity IL-13 receptors (Kd = 159 pM). These receptors are functional because IL-13 surprisingly increased proliferation of all three prostate cancer cell lines studied as determined by thymidine uptake and clonogenic assays. IL-13 receptors on prostate cancer cell lines were targeted using a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE38QQR). This molecule, termed IL13-PE38QQR, has been found cytotoxic to all three prostate cancer cell lines as determined by the inhibition of protein synthesis. The IC50 ranged between 1 nmol/l, to 15 nmol/l. These data were confirmed by clonogenic assays in which IL13-PE38QQR almost completely inhibited colony formation at 10 nmol/l. IL13-PE38QQR was not cytotoxic to cells that express little or no IL-13R. Heat inactivated IL13-PE38QQR was not cytotoxic to prostate cancer cells indicating specificity. IL13-PE38QQR was also cytotoxic to colonies when they were allowed to form first for several days before the addition of toxins. Our data suggest that additional studies should be performed to target IL-13 receptor bearing prostate cancer.

  20. Impaired atrial M(2)-cholinoceptor function in obesity-related hypertension.

    Science.gov (United States)

    Pelat, M; Verwaerde, P; Merial, C; Galitzky, J; Berlan, M; Montastruc, J L; Senard, J M

    1999-11-01

    The aim of this study was to investigate the activity of the parasympathetic limb of the baroreflex arch in a canine model of obesity-related hypertension. Twelve male beagle dogs were randomized into 2 groups. Six dogs were fed with normal canine food and 6 were submitted to a 10-week high-fat diet (HFD). We have evaluated the consequences of HFD on heart rate (HR) and blood pressure (BP) circadian cycles and methylscopolamine dose-response curves. Binding of [(3)H]-AF-DX 384 and adenylyl cyclase activity were investigated to determine the density and functionality of M(2)-cholinoceptors on right atrial membranes from control and HFD dogs. HFD induced a significant increase in body weight (15+/-1 vs 12+/-1 kg), systolic BP (161+/-5 vs 145+/-4 mm Hg), diastolic BP (92+/-3 vs 79+/-2 mm Hg), and HR (96+/-4 vs 81+/-3 bpm). Circadian rhythms of HR and BP observed in the baseline period were abolished after 9 weeks of HFD. After propranolol (1 mg/kg) pretreatment, the dose of methylscopolamine able to induce 50% maximum tachycardia was significantly increased after 9 weeks of HFD (7.4+/-0.3 vs 4.7+/-0.1 microg/kg). In the control group, the experimental period failed to modify these parameters. The numbers of M(2)-cholinoceptors measured in right atrial membranes were significantly lower in HFD than in control groups (54+/-6 vs 27+/-6 fmol/mg protein). The ability of carbachol to inhibit isoproterenol-stimulated adenylyl cyclase activity was significantly lower in HFD than in control groups (IC(50)=47+/-12 vs 6.4+/-1.4 micromol/L). However, the basal activity of adenylyl cyclase was unchanged by HFD. HFD decreases M(2)-cholinoceptor number and function in cardiomyocytes. This could explain the abolition of circadian rhythm of HR and the changes in chronotropic effect brought about by methylscopolamine.

  1. 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.

  2. Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5.

    Science.gov (United States)

    Wald, Tomas; Osickova, Adriana; Sulc, Miroslav; Benada, Oldrich; Semeradtova, Alena; Rezabkova, Lenka; Veverka, Vaclav; Bednarova, Lucie; Maly, Jan; Macek, Pavel; Sebo, Peter; Slaby, Ivan; Vondrasek, Jiri; Osicka, Radim

    2013-08-02

    Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.