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Sample records for forms protein body-like

  1. Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

    Science.gov (United States)

    Horiguchi, Ayumi; Yazaki, Koyuki; Aoyagi, Mami; Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

    2014-11-01

    Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Detergent Insoluble Proteins and Inclusion Body-Like Structures Immunoreactive for PRKDC/DNA-PK/DNA-PKcs, FTL, NNT, and AIFM1 in the Amygdala of Cognitively Impaired Elderly Persons.

    Science.gov (United States)

    Gal, Jozsef; Chen, Jing; Katsumata, Yuriko; Fardo, David W; Wang, Wang-Xia; Artiushin, Sergey; Price, Douglas; Anderson, Sonya; Patel, Ela; Zhu, Haining; Nelson, Peter T

    2018-01-01

    Misfolded protein in the amygdala is a neuropathologic feature of Alzheimer disease and many other neurodegenerative disorders. We examined extracts from human amygdala (snap-frozen at autopsy) to investigate whether novel and as yet uncharacterized misfolded proteins would be detectable. Polypeptides from the detergent-insoluble, urea-soluble protein fractions of amygdala were interrogated using liquid chromatography-electrospray ionization-tandem mass spectrometry. Among the detergent-insoluble proteins identified in amygdala of demented subjects but not controls were Tau, TDP-43, Aβ, α-synuclein, and ApoE. Additional detergent-insoluble proteins from demented subjects in the high-molecular weight portion of SDS gels included NNT, TNIK, PRKDC (DNA-PK, or DNA-PKcs), ferritin light chain (FTL), AIFM1, SYT11, STX1B, EAA1, COL25A1, M4K4, CLH1, SQSTM, SYNJ1, C3, and C4. In follow-up immunohistochemical experiments, NNT, TNIK, PRKDC, AIFM1, and FTL were observed in inclusion body-like structures in cognitively impaired subjects' amygdalae. Double-label immunofluorescence revealed that FTL and phospho-PRKDC immunoreactivity colocalized partially with TDP-43 and/or Tau inclusion bodies. Western blots showed high-molecular weight "smears", particularly for NNT and PRKDC. A preliminary genetic association study indicated that rare NNT, TNIK, and PRKDC gene variants had nominally significant association with Alzheimer-type dementia risk. In summary, novel detergent-insoluble proteins, with evidence of proteinaceous deposits, were found in amygdalae of elderly, cognitively impaired subjects. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

  3. Identifying proteins that can form tyrosine-cysteine crosslinks.

    Science.gov (United States)

    Martinie, Ryan J; Godakumbura, Pahan I; Porter, Elizabeth G; Divakaran, Anand; Burkhart, Brandon J; Wertz, John T; Benson, David E

    2012-10-01

    Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

  4. The CTCF insulator protein forms an unusual DNA structure

    Directory of Open Access Journals (Sweden)

    Sadowski Paul D

    2010-12-01

    Full Text Available Abstract Background The CTCF insulator protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. The protein has been hypothesized to organize the human genome by forming DNA loops. Results In this paper, we report biochemical evidence to support the role for CTCF in forming DNA loops. We have measured DNA bending by CTCF at the chicken HS4 β-globin FII insulator element in vitro and have observed a unique DNA structure with aberrant electrophoretic mobility which we believe to be a DNA loop. CTCF is able to form this unusual DNA structure at two other binding sites: the c-myc P2 promoter and the chicken F1 lysozyme gene silencer. We also demonstrate that the length though not the sequence of the DNA downstream of the binding site is important for the ability of CTCF to form this unusual DNA structure. We hypothesize that a single CTCF protein molecule is able to act as a "looper" possibly through the use of several of its zinc fingers. Conclusions CTCF is able to form an unusual DNA structure through the zinc finger domain of the protein. This unusual DNA structure is formed in a directional manner by the CTCF protein. The findings described in this paper suggest mechanisms by which CTCF is able to form DNA loops, organize the mammalian genome and function as an insulator protein.

  5. Reactive species formed on proteins exposed to singlet oxygen

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2004-01-01

    hydroperoxides, which can be reduced to the corresponding alcohols; other products arising from radical intermediates can also be generated, particularly in the presence of UV light and metal ions. With His side-chains, poorly characterised peroxides are also formed. Reaction with Met and Cys has been proposed...... to occur via zwitterionic peroxy intermediates. Peroxides are also generated on isolated proteins, and protein within intact cells, via(1)O(2)-mediated reactions. The peroxides formed on Trp, Tyr, and His peptides, as well as on proteins, have been shown to induce damage to other targets, with molecular...... oxidation of thiol residues an important reaction. This can result in the inactivation of cellular enzymes and the oxidation of other biological targets. Protein cross-linking and aggregation can also be induced by reactive species formed on photo-oxidised proteins, though the nature of the species...

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

    Science.gov (United States)

    Janner, A.

    2008-07-01

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

  7. Neighborhood Walkable Urban Form and C-Reactive Protein

    Science.gov (United States)

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

  8. The PIN-FORMED (PIN) protein family of auxin transporters.

    Science.gov (United States)

    Krecek, Pavel; Skupa, Petr; Libus, Jirí; Naramoto, Satoshi; Tejos, Ricardo; Friml, Jirí; Zazímalová, Eva

    2009-01-01

    The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.

  9. Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Maňásková, Pavla; Tichá, M.; Jonáková, Věra

    2003-01-01

    Roč. 32, - (2003), s. 99-107 ISSN 0141-8130 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : proteinase inhibitors, aggregated forms, boar seminal plasma proteins Subject RIV: CE - Biochemistry Impact factor: 1.427, year: 2003

  10. 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.

  11. Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

    Science.gov (United States)

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

    2014-01-01

    Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions. PMID:25463627

  12. Analysis of nanoparticle-protein coronas formed in vitro between nanosized welding particles and nasal lavage proteins.

    OpenAIRE

    Ali, Neserin; Mattsson, Karin; Rissler, Jenny; Karlsson, Helen Marg; Svensson, Christian R; Gudmundsson, Anders; Lindh, Christian; Jönsson, Bo A; Cedervall, Tommy; Kåredal, Monica

    2015-01-01

    Abstract Welding fumes include agglomerated particles built up of primary nanoparticles. Particles inhaled through the nose will to some extent be deposited in the protein-rich nasal mucosa, and a protein corona will be formed around the particles. The aim was to identify the protein corona formed between nasal lavage proteins and four types of particles with different parameters. Two of the particles were formed and collected during welding and two were manufactured iron oxides. When nasal l...

  13. 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......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...

  14. 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...

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

    Science.gov (United States)

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

    2017-07-24

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

  16. 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.

  17. Sampling Protein Form and Function with the Atomic Force Microscope

    NARCIS (Netherlands)

    Baclayon, Marian; Roos, Wouter H.; Wuite, Gijs J. L.

    To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e. g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe

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

    African Journals Online (AJOL)

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

  19. The PIN-FORMED (PIN) protein family of auxin transporters

    Czech Academy of Sciences Publication Activity Database

    Křeček, Pavel; Skůpa, Petr; Libus, Jiří; Naramoto, S.; Tejos, R.; Friml, J.; Zažímalová, Eva

    2009-01-01

    Roč. 10, č. 12 (2009), s. 249.1-249.11 ISSN 1474-760X R&D Projects: GA MŠk(CZ) LC06034; GA AV ČR KJB600380904; GA AV ČR(CZ) IAA601630703 Institutional research plan: CEZ:AV0Z50380511 Keywords : PIN protein family * auxin efflux carriers * auxin transport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.626, year: 2009

  20. Removal of maxillary sinus metallic foreign body like a hand sewing needle by magnetic iron.

    Science.gov (United States)

    Shao, Linqin; Qin, Xiurong; Ma, Yingwei

    2014-01-01

    Metallic foreign bodies are rarely found in the maxillary sinus, and usually they have a dental origin. Two main surgical app-roaches are currently used for the removal of foreign bodies in the maxillary sinus: the bone flap and the endoscopic sinus tech-niques. However, the treatment is not only surgical removal. We are reporting one case of foreign body like a hand sewing needle entered into the maxillary sinus through an unusual route- carious deciduous molar tooth. It was diagnosed by three-dimensional images from cone-beam computed tomography (CBCT) and removed by a simple procedure, with magnetic iron, thereby avoiding the risk of damage to a large portion of the alveolar bone near the maxillary sinus. How to cite this article: Shao L, Qin X, Ma Y. Removal of Maxillary Sinus Metallic Foreign Body Like a Hand Sewing Needle by Magnetic Iron. Int J Clin Pediatr Dent 2014;7(1):61-64.

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

    African Journals Online (AJOL)

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

  2. Origins and evolution of the HET-s prion-forming protein: searching for other amyloid-forming solenoids.

    Directory of Open Access Journals (Sweden)

    Deena M A Gendoo

    Full Text Available The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has "limited scope" for amyloidosis across the wider protein universe, compared to the 'generic' left-handed beta helix. We discuss the

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  4. 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

  5. Single Particle Tracking to Characterize the Mechanism of Pore Formation by Pore Forming Proteins

    OpenAIRE

    Subburaj, Yamunadevi

    2014-01-01

    Pore formation is a common natural mechanism occurring in large number of organisms where proteins are involved as toxins, effectors in immune response or apoptosis. Despite intense research, the structural and dynamic aspects of oligomerization and membrane permeabilization by pore forming proteins remains poorly understood. In this work we have aimed to provide a better understanding on dynamics, oligomerization and pore forming process of two proteins; a) Equinatoxin II, b) Bax (Bcl2 famil...

  6. Analysis of nanoparticle–protein coronas formed in vitro between nanosized welding particles and nasal lavage proteins

    Science.gov (United States)

    Ali, Neserin; Mattsson, Karin; Rissler, Jenny; Karlsson, Helen Marg; Svensson, Christian R.; Gudmundsson, Anders; Lindh, Christian H.; Jönsson, Bo A. G.; Cedervall, Tommy; Kåredal, Monica

    2016-01-01

    Abstract Welding fumes include agglomerated particles built up of primary nanoparticles. Particles inhaled through the nose will to some extent be deposited in the protein-rich nasal mucosa, and a protein corona will be formed around the particles. The aim was to identify the protein corona formed between nasal lavage proteins and four types of particles with different parameters. Two of the particles were formed and collected during welding and two were manufactured iron oxides. When nasal lavage proteins were added to the particles, differences were observed in the sizes of the aggregates that were formed. Measurements showed that the amount of protein bound to particles correlated with the relative size increase of the aggregates, suggesting that the surface area was associated with the binding capacity. However, differences in aggregate sizes were detected when nasal proteins were added to UFWF and Fe2O3 particles (having similar agglomerated size) suggesting that yet parameters other than size determine the binding. Relative quantitative mass spectrometric and gel-based analyses showed differences in the protein content of the coronas. High-affinity proteins were further assessed for network interactions. Additional experiments showed that the inhibitory function of secretory leukocyte peptidase inhibitor, a highly abundant nasal protein, was influenced by particle binding suggesting that an understanding of protein function following particle binding is necessary to properly evaluate pathophysiological events. Our results underscore the importance of including particles collected from real working environments when studying the toxic effects of particles because these effects might be mediated by the protein corona. PMID:26186033

  7. Analysis of nanoparticle-protein coronas formed in vitro between nanosized welding particles and nasal lavage proteins.

    Science.gov (United States)

    Ali, Neserin; Mattsson, Karin; Rissler, Jenny; Karlsson, Helen Marg; Svensson, Christian R; Gudmundsson, Anders; Lindh, Christian H; Jönsson, Bo A G; Cedervall, Tommy; Kåredal, Monica

    2016-01-01

    Welding fumes include agglomerated particles built up of primary nanoparticles. Particles inhaled through the nose will to some extent be deposited in the protein-rich nasal mucosa, and a protein corona will be formed around the particles. The aim was to identify the protein corona formed between nasal lavage proteins and four types of particles with different parameters. Two of the particles were formed and collected during welding and two were manufactured iron oxides. When nasal lavage proteins were added to the particles, differences were observed in the sizes of the aggregates that were formed. Measurements showed that the amount of protein bound to particles correlated with the relative size increase of the aggregates, suggesting that the surface area was associated with the binding capacity. However, differences in aggregate sizes were detected when nasal proteins were added to UFWF and Fe2O3 particles (having similar agglomerated size) suggesting that yet parameters other than size determine the binding. Relative quantitative mass spectrometric and gel-based analyses showed differences in the protein content of the coronas. High-affinity proteins were further assessed for network interactions. Additional experiments showed that the inhibitory function of secretory leukocyte peptidase inhibitor, a highly abundant nasal protein, was influenced by particle binding suggesting that an understanding of protein function following particle binding is necessary to properly evaluate pathophysiological events. Our results underscore the importance of including particles collected from real working environments when studying the toxic effects of particles because these effects might be mediated by the protein corona.

  8. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    Directory of Open Access Journals (Sweden)

    Weipeng eZhang

    2015-05-01

    Full Text Available Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm.

  9. Developmental-form-specific DNA-binding proteins in Chlamydia spp.

    Science.gov (United States)

    Wagar, E A; Stephens, R S

    1988-01-01

    We identified DNA-binding proteins specific to the elementary body (EB) developmental form of Chlamydia spp. Chlamydial proteins from whole lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with nick-translated chlamydial DNA. By this method, C. trachomatis serovar L2 EBs had three unique protein bands of 58,000, 25,700, and 17,000 molecular weight not seen in the reticulate bodies. The 17,000-molecular-weight protein and the 25,700-molecular-weight protein were identified in an acid-soluble protein fraction and were resistant to high-salt elution, similar to other procaryotic nucleoproteins. The 17,000-molecular-weight protein was also detected in preparations with isolated chromosomes from EBs. Preliminary characterization indicated that the protein-DNA interaction was principally charge related. Images PMID:3384472

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

    Czech Academy of Sciences Publication Activity Database

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

    2004-01-01

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

  11. 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

  12. The effect of concentrate in meal- or pellet form on milk, protein and ...

    African Journals Online (AJOL)

    sources, on milk, protein and butterfat production of. Friesian cows was ... consume concentrate pellets much quicker than concentrate meal. The main ... Total protein kg milk produced. Days. Form of production per production per production per per concentrate. Phase. Group measured concentrate cow (kg) cow (kg).

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

    NARCIS (Netherlands)

    Domeradzka, N.E.; Werten, M.W.T.; Vries, de R.J.; Wolf, de F.A.

    2016-01-01

    Some combinations of leucine zipper peptides are capable of forming a-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

  14. 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

    by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo...

  15. Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation.

    Science.gov (United States)

    Arvanitis, Demetrios A; Vafiadaki, Elizabeth; Papalouka, Vasiliki; Sanoudou, Despina

    2017-12-01

    Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca 2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Brann Jessica H

    2010-05-01

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

  17. Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Hassan Ramshini

    Full Text Available The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i low pH with salts, (ii pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

  18. Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification

    International Nuclear Information System (INIS)

    Ingoglia, N.A.; Chakroborty, G.; Yu, M.; Luo, D.; Sturman, J.A.

    1991-01-01

    Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates

  19. Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins.

    Science.gov (United States)

    Serricchio, Mauro; Vissa, Adriano; Kim, Peter K; Yip, Christopher M; McQuibban, G Angus

    2018-04-01

    The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. 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.

  1. UV-light exposed prion protein fails to form amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Abhay Kumar Thakur

    Full Text Available Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.

  2. 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

  3. Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells.

    Science.gov (United States)

    Abrahám, Edit; Miskolczi, Pál; Ayaydin, Ferhan; Yu, Ping; Kotogány, Edit; Bakó, László; Otvös, Krisztina; Horváth, Gábor V; Dudits, Dénes

    2011-03-01

    Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.

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

    OpenAIRE

    Macrander, Jason; Daly, Marymegan

    2016-01-01

    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...

  5. Rpf Proteins Are the Factors of Reactivation of the Dormant Forms of Actinobacteria.

    Science.gov (United States)

    Nikitushkin, V D; Demina, G R; Kaprelyants, A S

    2016-12-01

    As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its participation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis - an infectious disease that affects one third of the World's population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine - in diagnostics and in creation of antituberculosis subunit vaccines.

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

    Science.gov (United States)

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

    2015-12-28

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    International Nuclear Information System (INIS)

    Sabato, Susy Frey

    2000-01-01

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

  10. 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...

  11. Data on structural transitions in domains of hordeivirus TGB1 protein forming ribonucleoprotein complex

    Directory of Open Access Journals (Sweden)

    Valentin V. Makarov

    2016-09-01

    Full Text Available This data article is related to the research article entitled “in vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes” (Makarov et al., 2015 [1], demonstrating that upon incubation with viral RNA the poa semilatent hordeivirus (PSLV TGB1 protein (the movement 63 K protein encoded by the first gene of the triple gene block in vitro forms RNP structures resembling filamentous virus-like particles and its internal domain (ID performs a major structural role in this process. This article reports the additional results on the structural lability of ID and the structural transitions in the C-terminal NTPase/helicase domain (HELD induced by interaction with tRNA and phosphorylation.

  12. A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

    Science.gov (United States)

    Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

    2016-02-01

    We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. The Oligomeric Form of the Escherichia coli Dps Protein Depends on the Availability of Iron Ions

    Directory of Open Access Journals (Sweden)

    Sergey Antipov

    2017-11-01

    Full Text Available The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr’s salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe3O4, rather than the expected Fe2O3, in the sample. Assuming that Fe2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe2O3 with Dps, but the binding sites with putative functionality were predicted only for Fe2O3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.

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

    Science.gov (United States)

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

    1999-01-08

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

  15. Comparative methods for analysis of protein covalent modification by electrophilic quinoids formed from xenobiotics

    Science.gov (United States)

    Yu, Bolan; Qin, Zhihui; Wijewickrama, Gihani T.; Edirisinghe, Praneeth; Bolton, Judy L.; Thatcher, Gregory R. J.

    2010-01-01

    Conjugation of biotin and fluorophore tags is useful for assaying covalent protein modification. Oxidative bioactivation of selective estrogen receptor modulators (SERMs) yields reactive quinoid electrophiles that covalently modify proteins; bioactivation is associated with carcinogenic and chemopreventive effects. Identification of the protein targets of electrophilic metabolites is of general important for xenobiotics. Four methodologies, using SERM derivatives and biotin/fluorophore tags, were compared for purification and quantification: (1) covert oxidatively activated tags (COATags; SERM conjugated to biotin); (2) dansylTags (SERM conjugated to fluorophore); and azidoTags (SERM azide derivatives) in a 2-step conjugation to biotin, either using (3) Staudinger ligation; or (4) click chemistry. All synthetic derivatives retained the estrogen receptor ligand characteristics of the parent SERMs. Model proteins with bioactivation by tyrosinase in buffer or cell lysates and liver proteins with in situ bioactivation in rat primary hepatocytes were studied by immunoassay and fluorescence. Comparison showed: the azidoTag/Staudinger method was sensitive but nonspecific; the azidoTag/click methodology had low sensitivity; and, the dansylTag methodology failed to detect modified proteins in hepatocytes. The COATag methodology was adjudged superior, detecting 5 ng of modified protein in vitro and identifying protein targets in hepatocytes. In metabolism studies in rat liver microsomes, the azide group was metabolically labile, one contributing factor in not selecting an azidoTag methodology in the highly oxidative environments required for bioactivation. For study of the protein targets of electrophilic metabolites formed by in situ oxidative bioactivation, the COATag is both sensitive and specific, and does not appear to suffer from poor cell permeability. PMID:19301905

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

    Science.gov (United States)

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

    2018-04-01

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

  17. 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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  19. Organ distribution and molecular forms of human xanthine dehydrogenase/xanthine oxidase protein.

    Science.gov (United States)

    Sarnesto, A; Linder, N; Raivio, K O

    1996-01-01

    Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmic source of superoxide radicals and hydrogen peroxide, and it is considered important in the pathogenesis of ischemia-reperfusion damage. Because little is known about the enzyme in human tissues, the aims of this study were to purify human XDH/XO and to produce Ab for detection of the protein in Western blots and for quantification by ELISA. We purified human milk XDH/XO, produced Ab for Western blotting and ELISA of the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified protein under nondenaturing conditions was approximately 300 kd. On SDS-PAGE, it was fragmented into four main bands of 143, 125, 87, and 59 kd. Ab recognized bands of similar size in Western blots of the purified preparation and human milk. In fresh liver homogenates treated with anti-proteases, the three largest bands were observed; in the intestine, only the two largest were observed. Serum, brain, heart, and skeletal muscle were negative, whereas some lung and kidney samples showed one faint band of 143 kd. Trypsin treatment of the enzyme converted the large molecular-weight bands into smaller bands, as did incubation of a liver homogenate without anti-proteases. XDH/XO protein concentrations (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in intestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mumol/min/mg XDH/XO protein) was constant in liver and intestine during development. We conclude that 1) human XDH/XO has molecular size and subunit structure similar to other mammalian enzymes; 2) the polypeptide chain is unstable, also in the intact cell, despite retained activity; and 3) the amount of inactive XDH/XO in human liver and intestine is apparently small.

  20. 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.

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

    International Nuclear Information System (INIS)

    Galat, Andrzej; Thai, Robert

    2014-01-01

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

  2. Stress induced nuclear granules form in response to accumulation of misfolded proteins in Caenorhabditis elegans.

    Science.gov (United States)

    Sampuda, Katherine M; Riley, Mason; Boyd, Lynn

    2017-04-19

    Environmental stress can affect the viability or fecundity of an organism. Environmental stressors may affect the genome or the proteome and can cause cellular distress by contributing to protein damage or misfolding. This study examines the cellular response to environmental stress in the germline of the nematode, C. elegans. Salt stress, oxidative stress, and starvation, but not heat shock, induce the relocalization of ubiquitin, proteasome, and the TIAR-2 protein into distinct subnuclear regions referred to as stress induced nuclear granules (SINGs). The SINGs form within 1 h of stress initiation and do not require intertissue signaling. K48-linked polyubiquitin chains but not K63 chains are enriched in SINGs. Worms with a mutation in the conjugating enzyme, ubc-18, do not form SINGs. Additionally, knockdown of ubc-20 and ubc-22 reduces the level of SING formation as does knockdown of the ubiquitin ligase chn-1, a CHIP homolog. The nuclear import machinery is required for SING formation. Stressed embryos containing SINGs fail to hatch and cell division in these embryos is halted. The formation of SINGs can be prevented by pre-exposure to a brief period of heat shock before stress exposure. Heat shock inhibition of SINGs is dependent upon the HSF-1 transcription factor. The heat shock results suggest that chaperone expression can prevent SING formation and that the accumulation of damaged or misfolded proteins is a necessary precursor to SING formation. Thus, SINGs may be part of a novel protein quality control system. The data suggest an interesting model where SINGs represent sites of localized protein degradation for nuclear or cytosolic proteins. Thus, the physiological impacts of environmental stress may begin at the cellular level with the formation of stress induced nuclear granules.

  3. 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.

  4. 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

    2016-10-01

    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. © 2016 The Author(s).

  5. 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.

  6. Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Walian, P.J.

    1989-11-01

    One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing of these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)

  7. 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

  8. G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.

    Science.gov (United States)

    Lavine, Natalie; Ethier, Nathalie; Oak, James N; Pei, Lin; Liu, Fang; Trieu, Phan; Rebois, R Victor; Bouvier, Michel; Hebert, Terence E; Van Tol, Hubert H M

    2002-11-29

    A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    2008-07-01

    form of the prion protein ( PrPSc ). However, there is mounting evidence that PrPSc is not directly toxic to neurons; it may require interaction with...Hegde et al. 1999]. This hypothesis suggests that 4 PrPSc itself is not toxic per se, but it causes disease by leading to the aberrant generation of...However, this does not rule out the separate hypothesis that CtmPrP and PrPSc may be separately activating a common neurodegenerative pathway

  11. Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

    OpenAIRE

    Bhasin, R; Van Nostrand, W E; Saitoh, T; Donets, M A; Barnes, E A; Quitschke, W W; Goldgaber, D

    1991-01-01

    Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and sho...

  12. 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

    1996-07-01

    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 can 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.

  13. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  15. 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.

  16. 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.

  17. 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. PMID:23612307

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    M. Lauren Donnelly

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Victor M Aguilera

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    Kruse, Torben; Bork-Jensen, J.; Gerdes, Kenn

    2005-01-01

    . subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became......D. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based......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...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-06-04

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

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

    Science.gov (United States)

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

    2017-11-01

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

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Angela Schwede

    2015-12-01

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

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

    Science.gov (United States)

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

    2016-08-01

    The fluorescent protein from Dendronephthya sp. (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 C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond 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.

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

    International Nuclear Information System (INIS)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

    A large number of collagen-like proteins have been identified in bacteria during the past ten years, 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 E. 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. PMID:24434612

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

    Science.gov (United States)

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

    1997-02-01

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

  12. Freeze-drying of proteins in glass solids formed by basic amino acids and dicarboxylic acids.

    Science.gov (United States)

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

    2009-01-01

    The purpose of this study was to produce and characterize glass-state amorphous solids containing amino acids and organic acids that protect co-lyophilized proteins. Thermal analysis of frozen solutions containing a basic amino acid (e.g., L-arginine, L-lysine, L-histidine) and a hydroxy di- or tricarboxylic acid (e.g., citric acid, L-tartaric acid, DL-malic acid) showed glass transition of maximally freeze-concentrated solute at temperatures (T'g) significantly higher than those of the individual solute solutions. Mixing of the amino acid with some dicarboxylic acids (e.g., oxalic acid) also suggested an upward shift of the transition temperature. Contrarily, combinations of the amino acid with monocarboxylic acids (e.g., acetic acid) had T'gs between those of the individual solute solutions. Co-lyophilization of the basic amino acids and citric acid or L-tartaric acid resulted in amorphous solids that have glass transition temperatures (Tg) higher than the individual components. Mid- and near-infrared analysis indicated altered environment around the functional groups of the consisting molecules. Some of the glass-state excipient combinations protected an enzyme (lactate dehydrogenase, LDH) from inactivation during freeze-drying. The glass-state excipient combinations formed by hydrogen-bonding and electrostatic interaction network would be potent alternative to stabilize therapeutic proteins in freeze-dried formulations.

  13. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  15. 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.

  16. 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.

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

    International Nuclear Information System (INIS)

    Smallwood, Sherin; Moyer, Sue A.

    2004-01-01

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

  18. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

    Science.gov (United States)

    Mackinder, Luke C M; Meyer, Moritz T; Mettler-Altmann, Tabea; Chen, Vivian K; Mitchell, Madeline C; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C

    2016-05-24

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

  19. 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.

  20. A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis.

    Science.gov (United States)

    Zhang, Maolei; Huang, Nunu; Yang, Xuesong; Luo, Jingyan; Yan, Sheng; Xiao, Feizhe; Chen, Wenping; Gao, Xinya; Zhao, Kun; Zhou, Huangkai; Li, Ziqiang; Ming, Liu; Xie, Bo; Zhang, Nu

    2018-01-18

    Circular RNAs (circRNAs) are recognized as functional non-coding transcripts in eukaryotic cells. Recent evidence has indicated that even though circRNAs are generally expressed at low levels, they may be involved in many physiological or pathological processes, such as gene regulation, tissue development and carcinogenesis. Although the 'microRNA sponge' function is well characterized, most circRNAs do not contain perfect trapping sites for microRNAs, which suggests the possibility that circRNAs have functions that have not yet been defined. In this study, we show that a circRNA containing an open reading frame (ORF) driven by the internal ribosome entry site (IRES) can translate a functional protein. The circular form of the SNF2 histone linker PHD RING helicase (SHPRH) gene encodes a novel protein that we termed SHPRH-146aa. Circular SHPRH (circ-SHPRH) uses overlapping genetic codes to generate a 'UGA' stop codon, which results in the translation of the 17 kDa SHPRH-146aa. Both circ-SHPRH and SHPRH-146aa are abundantly expressed in normal human brains and are down-regulated in glioblastoma. The overexpression of SHPRH-146aa in U251 and U373 glioblastoma cells reduces their malignant behavior and tumorigenicity in vitro and in vivo. Mechanistically, SHPRH-146aa protects full-length SHPRH from degradation by the ubiquitin proteasome. Stabilized SHPRH sequentially ubiquitinates proliferating cell nuclear antigen (PCNA) as an E3 ligase, leading to inhibited cell proliferation and tumorigenicity. Our findings provide a novel perspective regarding circRNA function in physiological and pathological processes. Specifically, SHPRH-146aa generated from overlapping genetic codes of circ-SHPRH is a tumor suppressor in human glioblastoma.

  1. Brain expressed and X-linked (Bex proteins are intrinsically disordered proteins (IDPs and form new signaling hubs.

    Directory of Open Access Journals (Sweden)

    Eva M Fernandez

    Full Text Available Intrinsically disordered proteins (IDPs are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex proteins consists of 5 members in humans (Bex1-5. Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in E. coli. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs.

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

    Science.gov (United States)

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

    2017-11-09

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

  3. Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Jung-Hyuck Ahn

    Full Text Available Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A. A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

  4. Flicker downregulates the content of crystallin proteins in form-deprived C57BL/6 mouse retina.

    Science.gov (United States)

    Li, Saiqun; Wu, Junshu; Ding, Hui; Liao, Aiping; He, Hong; Stell, William K; Zhong, Xingwu

    2012-08-01

    Image degradation by loss of higher spatial frequencies causes form-deprivation myopia (FDM) in humans and animals, and cyclical illumination (flicker) at certain frequencies may prevent FDM. The molecular mechanisms underlying FDM and its prevention by flicker are poorly known. To understand them better, we have identified proteins that differ in amount in form-deprived (FD) mouse retinas, under steady versus flickering light. Male C57BL/6 mice (age 27-29 days) were randomly divided into three groups: Experimental - monocularly form-deprived, and kept under either normal room light ("FD-Only") or 20 Hz flickering light ("FD-Flicker"), throughout the 12-hour light phase; and Control ("Open-Control") - kept under normal illumination, without form deprivation. After two weeks of treatment, retinal proteins were extracted and separated by two-dimensional gel electrophoresis (2D-GE); proteins that differ in content in FD-only versus FD-flicker retinas were identified by mass spectroscopy ("MS"), and their identities were verified by western blotting. The contents of three identified proteins differed statistically in FD-only compared to FD-flicker retinas. These proteins were identified by MS as α-A-crystallin, crystallin β A2 and crystallin β A1. Quantitative western blotting showed that the relative amount of α-A-crystallin in FD-only retinas was significantly higher than that in FD-Flicker and control retinas. In conclusion, form deprivation induced significant increases in the amounts of crystallins in mouse retinas. These increases were significantly reduced by exposure to 20 Hz flicker. Since form deprivation is known to induce myopia development, and flicker to prevent it, our data suggest that FD- and flicker-responsive changes in the content of crystallin proteins may be involved causally or protectively in myopia development. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Structure and dynamics of the membrane-bound form of the filamentous bacteriophage coat proteins by NMR spectroscopy

    International Nuclear Information System (INIS)

    Bogusky, M.J.

    1987-01-01

    The structure and dynamics of the Pf1 and fd bacteriophage coat proteins in detergent micelles are characterized in solution by nuclear magnetic resonance spectroscopy. The coat proteins are found to exist within the bacterial inner cell membrane during viral infection and assembly. The coat proteins serve as a model system to investigate integral membrane proteins as well as the viral infection and assembly processes. The coat protein is insoluble in aqueous or organic solvents and can only be effectively solubilized in the presence of detergents that form micelles or phospholipids that form vesicles. The effective molecular weight of the detergent-micelle complex is ca. 30K daltons. Sequential assignment strategies were ineffective due to short T/sub 2s/ and severe resonance degeneracy. The backbone resonance assignments were completed by the combination of several homo- and heteronuclear correlation techniques with biosynthetic 15 N labelling. 2D NOE experiments were used to locate and characterize the secondary structure of the membrane bound form of the proteins showing them to be largely helical with the hydrophobic core existing in a very stable helix

  6. Multimers Formed by the Rotavirus Nonstructural Protein NSP2 Bind to RNA and Have Nucleoside Triphosphatase Activity

    OpenAIRE

    Taraporewala, Zenobia; Chen, Dayue; Patton, John T.

    1999-01-01

    The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 ± 2 subuni...

  7. 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.

  8. Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

    Science.gov (United States)

    Bounedjah, Ouissame; Desforges, Bénédicte; Wu, Ting-Di; Pioche-Durieu, Catherine; Marco, Sergio; Hamon, Loic; Curmi, Patrick A; Guerquin-Kern, Jean-Luc; Piétrement, Olivier; Pastré, David

    2014-07-01

    The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. 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...

  10. 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

  11. Characterization of Truncated Forms of Abnormal Prion Protein in Creutzfeldt-Jakob Disease

    NARCIS (Netherlands)

    Notari, S.; Strammiello, R.; Capellari, S.; Giese, A.; Cescatti, M.; Grassi, J.; Ghetti, B.; Langeveld, J.P.M.; Zou, W.Q.; Gambetti, P.; Kretzschmar, H.A.; Parchi, P.

    2008-01-01

    In prion disease, the abnormal conformer of the cellular prion protein, PrPSc, deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrPSc to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent

  12. 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

  13. Immunogenicity studies of proteins forming the T4 phage head surface.

    Science.gov (United States)

    Dąbrowska, Krystyna; Miernikiewicz, Paulina; Piotrowicz, Agnieszka; Hodyra, Katarzyna; Owczarek, Barbara; Lecion, Dorota; Kaźmierczak, Zuzanna; Letarov, Andrey; Górski, Andrzej

    2014-11-01

    Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages. Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere

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

    OpenAIRE

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

    2015-01-01

    Abstract Background Transporter proteins are predicted to have an important role in the mycorrhizal symbiosis, due to the fact that this type of an interaction between plants and fungi requires a continuous nutrient and signalling exchange. ABC transporters are one of the large groups of transporter proteins found both in plants and in fungi. The crucial role of plant ABC transporters in the formation of the mycorrhizal symbiosis has bee...

  15. Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

    OpenAIRE

    Flores, Francisco J; Martín, Juan F

    2004-01-01

    In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes form...

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

    OpenAIRE

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

    2010-01-01

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

  17. Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in theYersinia enterocoliticaCytoplasmic Membrane.

    Science.gov (United States)

    Srivastava, Disha; Moumene, Amal; Flores-Kim, Josué; Darwin, Andrew J

    2017-09-12

    The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of

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

    Science.gov (United States)

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

    2010-07-01

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

  19. 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.

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

    Science.gov (United States)

    Diaz-Espinoza, Rodrigo; Mukherjee, Abhisek; Soto, Claudio

    2012-01-01

    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.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  3. 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

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

    Directory of Open Access Journals (Sweden)

    Thiago Novaes Silva

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

  5. 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

  6. 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

    A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two...... porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations....

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

    Science.gov (United States)

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

    2018-03-03

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

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

    Science.gov (United States)

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

    1985-03-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  10. 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.

  11. Isolation of complexes formed between insulin-like growth factor-binding protein-3 and transferrin from the human serum

    Directory of Open Access Journals (Sweden)

    Miljuš Goran

    2012-01-01

    Full Text Available Insulin-like growth factors (IGFs play an important role in the regulation of cell growth, differentiation and metabolism. The amount of free, biologically active IGFs is regulated by the IGF-binding proteins (IGFBPs. IGFBP-3 is the most abundant binding protein and it is known to interact with other circulating proteins, including transferrin (Tf. In order to elucidate the possible role of IGF/IGFBP-3 in the iron metabolism, it is necessary to isolate IGFBP-3/Tf complexes. Several affinity-based techniques were employed. Results have shown that only double immunoprecipitation method with anti-Tf and anti-IGFBP-3 antibodies selectively separated complexes from other molecular forms, such as monomers, oligomers or fragments of IGFBP-3 and Tf. Isolated complexes can now be used to investigate the relationship between IGF/IGFBP-3 and iron, both in structural and metabolic tеrms.

  12. 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.

  13. 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.

    2012-01-01

    Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 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. PMID:22267004

  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

    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.

  16. Protein SIC Secreted fromForms Complexes with Extracellular Histones That Boost Cytokine Production

    DEFF Research Database (Denmark)

    Westman, Johannes; Chakrakodi, Bhavya; Snäll, Johanna

    2018-01-01

    determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein fromStreptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue...... damage. This interaction leads to the formation of large aggregatesin vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused byS. pyogenes. In addition, binding of SIC to histones neutralized...... their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines...

  17. The origins of life -- the 'protein interaction world' hypothesis: protein interactions were the first form of self-reproducing life and nucleic acids evolved later as memory molecules.

    Science.gov (United States)

    Andras, Peter; Andras, Csaba

    2005-01-01

    The 'protein interaction world' (PIW) hypothesis of the origins of life assumes that life emerged as a self-reproducing and expanding system of protein interactions. In mainstream molecular biology, 'replication' refers to the material copying of molecules such as nucleic acids. However, PIW is conceptualized as an abstract communication system constituted by the interactions between proteins, in which 'replication' happens at the level of self-reproduction of these interactions between proteins. Densely concentrated peptide interaction systems may have reproduced and expanded as 'protocell' vesicles surrounded by lipid bi-layer membranes. Protocells led to the emergence of proto-RNA molecules of greater chemical stability which served as chemically differentiated 'memories' of peptide interaction states, thereby facilitating the reproduction and expansion of protocells. Simplification-driven expansion led to the selection of biotic amino acids and the reduction of the typical RNA alphabet to the four usual bases (A, C, G and U). Dense interactions between RNA molecules led to the emergence of the RNA interaction subsystem of the cell, and to the emergence of 'memories' of RNA interactions in the form of DNA molecules with greater chemical stability. The expansion of DNA molecule interactions led to the dense clustering and encapsulation of DNA molecules within the cell nucleus. RNA molecules therefore serve as memories of protein interactions and DNA molecules are memories of RNA interactions. We believe that the PIW hypothesis is more evolutionarily plausible than the mainstream RNA world hypothesis, and has greater explanatory power.

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

    OpenAIRE

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

    1997-01-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), pro...

  19. Random protein sequences can form defined secondary structures and are well-tolerated in vivo

    Czech Academy of Sciences Publication Activity Database

    Tretyachenko, Vyacheslav; Vymětal, Jiří; Bednárová, Lucie; Kopecký, V. Jr.; Hofbauerová, K.; Jindrová, Helena; Hubálek, Martin; Souček, Radko; Konvalinka, Jan; Vondrášek, Jiří; Hlouchová, Klára

    2017-01-01

    Roč. 7, Nov 13 (2017), č. článku 15449. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : amino acid sequence * de novo proteins * structure prediction Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.259, year: 2016 https://www.nature.com/ articles /s41598-017-15635-8

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

    Czech Academy of Sciences Publication Activity Database

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

    2012-01-01

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

  1. Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

    DEFF Research Database (Denmark)

    Malacrida, Leonel; Astrada, Soledad; Briva, Arturo

    2016-01-01

    Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (lamellar body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process...

  2. A Long-Distance Translocatable Phloem Protein from Cucumber Forms a Ribonucleoprotein Complex In Vivo with Hop Stunt Viroid RNA†

    Science.gov (United States)

    Gómez, Gustavo; Pallás, Vicente

    2004-01-01

    Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed. PMID:15331743

  3. Integration of protein extraction with a stream of byproducts from marine macroalgae: A model forms the basis for marine bioeconomy.

    Science.gov (United States)

    Gajaria, Tejal K; Suthar, Poornima; Baghel, Ravi S; Balar, Nikunj B; Sharnagat, Preeti; Mantri, Vaibhav A; Reddy, C R K

    2017-11-01

    The present study describes an advanced biorefinery model for marine macroalgae that assumes significant importance in the context of marine bio-economy. The method investigated in this study integrates the extraction of crude proteins with recovery of minerals rich sap, lipids, ulvan and cellulose from fresh biomass of Ulva lactuca. The protein content extracted was 11±2.12% on dry weight basis with recovery efficiency of 68.75±4.01%. The amino acid composition of crude protein fraction showed iso-leucine as the most abundant amino acid with 16.51±0.03% followed by histidine, arginine, tyrosine, serine, aspartic acid, threonine, phenyl alanine, leucine, alanine, lysine, glycine and glutamic acid (0.22±0.24%). The digestibility of protein was as high as 85.86±5.92% indicating its suitability for use in food supplements. The protein production with co-recovery of other products would not only result in effective utilisation marine macroalgal resources but also forms the basis for marine bio-economy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Tunnel Formation Inferred from the I-Form Structures of the Proton-Driven Protein Secretion Motor SecDF

    Directory of Open Access Journals (Sweden)

    Arata Furukawa

    2017-05-01

    Full Text Available Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.

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

    Directory of Open Access Journals (Sweden)

    E. Yavuz

    2015-06-01

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

  6. 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.

  7. 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 o...... compared with F-actin, despite the similar arrangement of the subunits within the filaments. Thus, there is now evidence for cytoskeletal structures, formed by actin-like filaments that are involved in plasmid partitioning in E.coli. Udgivelsesdato: Dec 16...

  8. 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...

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

    Science.gov (United States)

    Barnea, G; Grumet, M; Milev, P; Silvennoinen, O; Levy, J B; Sap, J; Schlessinger, J

    1994-05-20

    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 immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system.

  10. Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro.

    Science.gov (United States)

    Antony, A C; Verma, R S

    1989-02-13

    Human erythrocyte membranes contain high-affinity folate-binding proteins (FBPs) which on solubilization with detergents resolve into apparent 160,000 Mr moieties on Sephacryl S-200 gel filtration in Triton X-100. These FBPs share antigenic and ligand binding characteristics with particulate FBPs from other human tissues. During studies to define the vectorial orientation of these FBPs on the erythrocytes, we trypsinized intact cells with 250 micrograms trypsin per ml packed cells and quantitatively analysed the remaining cell-associated FBPs as well as the products of proteolysed FBPs in the supernatant. Incubation of intact cells with trypsin resulted in a dose-dependent decrease in their capacity to bind 125I-labelled pteroylglutamate (histamine derivative); at 250 micrograms/ml trypsin, folate binding was decreased by 77% compared to nontrypsin-treated control cells. While trypsinized cells contained proportionately lower quantities of apparent 160,000 Mr FBPs than untreated control cells, the supernatant of trypsinized cells (soluble phase) contained a single species of Mr = 40,000 which retained folate binding capacity. The sum of FBPs in trypsin supernatant and trypsin-treated cells was 87% of that found in untreated cells. Analysis of solubilized particulate erythrocyte FBPs and soluble (trypsin product) FBPs by sucrose density gradient ultracentrifugation in H2O and 2H2O above the critical micellar concentration of Triton X-100 revealed that apparent 160,000 Mr FBPs were detergent-binding (hydrophobic) species (which sedimented at Mr = 40,000 in H2O) while soluble FBPs (also sedimenting at Mr = 40,000) were hydrophilic and did not bind Triton X-100. These are the first data which show that hydrophobic FBPs can be directly converted to hydrophilic FBPs by a trypsin-mediated proteolytic event. The trypsin-sensitive site is likely to be at the junction between the detergent-binding site and the major body of the protein (Mr = 40,000) containing the folate

  11. Pore-forming proteins with built-in triggers and switches

    Science.gov (United States)

    Bayley, Hagan

    1996-02-01

    Genetic engineering and targeted chemical modification are being used to produce polypeptides with pore-forming activity that can be triggered or switched on-and-off by biochemical, chemical or physical stimuli. The principal target of our studies has been the (alpha) -hemolysin ((alpha) HL) from the bacterium Staphylococcus aureus. The remodeled hemolysins include protease-activated pores, metal-regulated pores, pores that are activated by chemical alkylation and pores that are turned on with light. These polypeptides have several potential applications. For example, they might serve as components of sensors or they might be useful for mediating the controlled release of encapsulated drugs.

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

    Directory of Open Access Journals (Sweden)

    Sang Beum Lee

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

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

    Science.gov (United States)

    Lee, Sang Beum; Park, Sung Kwon; Kim, Yong Soo

    2017-01-01

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

  14. 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

    Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl...... 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...... protein....

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

    Science.gov (United States)

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

    2015-03-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Amyloid formation is associated with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including...... the molecular mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, we present experimental data based on hybrid analysis of two-photon-microscopy, solution...... secondary structure. As a result of membrane fragmentation, soluble αSN:-lipid coaggregates are formed, hence, suggesting a novel molecular mechanism behind PD amyloid cytotoxicity....

  18. 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.

  19. Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone.

    Science.gov (United States)

    Dohi, Yoshiko; Tabata, Shiro; Yamaguchi, Minoru; Ohgushi, Hajime; Yonemasu, Kunio

    2004-07-01

    A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).

  20. 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.

  1. Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity.

    Science.gov (United States)

    Taraporewala, Z; Chen, D; Patton, J T

    1999-12-01

    The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rNSP2). As shown by gel mobility shift assays, the rNSP2 multimers bound to RNA in discrete cooperative steps to form higher-order RNA-protein complexes. The RNA-binding activity of the rNSP2 multimers was determined to be nonspecific and to have a strong preference for single-stranded RNA over double-stranded RNA, for which it displayed little affinity. Enzymatic analysis revealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg(2+) catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicating that the hydrolysis of NTP resulted in the covalent linkage of the gamma-phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 expressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NTPase activity.

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

    Science.gov (United States)

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

    1994-01-01

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

  3. A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex.

    Science.gov (United States)

    Sivadas, Priyanka; Dienes, Jennifer M; St Maurice, Martin; Meek, William D; Yang, Pinfen

    2012-11-12

    A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH-RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.

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

    Directory of Open Access Journals (Sweden)

    Youn-Bok Lee

    2013-12-01

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  6. 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.

  7. P2X7R large pore is partially blocked by pore forming proteins antagonists in astrocytes.

    Science.gov (United States)

    Faria, Robson X; Reis, Ricardo A M; Ferreira, Leonardo G B; Cezar-de-Mello, Paula F T; Moraes, Milton O

    2016-06-01

    The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore.

  8. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

    Energy Technology Data Exchange (ETDEWEB)

    Murray, M.T.; Krohne, G.; Franke, W.W. (Institute of Cell and Tumor Biology, Heidelberg (Germany, F.R.))

    1991-01-01

    To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.

  9. Recognition of the Different Structural Forms of the Capsid Protein Determines the Outcome following Infection with Porcine Circovirus Type 2

    Science.gov (United States)

    Trible, Benjamin R.; Suddith, Andrew W.; Kerrigan, Maureen A.; Cino-Ozuna, Ada G.; Hesse, Richard A.

    2012-01-01

    Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169–180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169–180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43–233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169–180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169–180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169–180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection. PMID:23035215

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

    Directory of Open Access Journals (Sweden)

    Qing Wang

    2015-02-01

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

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

    International Nuclear Information System (INIS)

    Gonçalves, A. M. D.; Rêgo, A. T.; Thomaz, M.; Enguita, F. J.; Carrondo, M. A.

    2008-01-01

    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 1 2 1 2 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. 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.

  13. Playing RNase P evolution: swapping the RNA catalyst for a protein reveals functional uniformity of highly divergent enzyme forms.

    Directory of Open Access Journals (Sweden)

    Christoph Weber

    2014-08-01

    Full Text Available The RNase P family is a diverse group of endonucleases responsible for the removal of 5' extensions from tRNA precursors. The diversity of enzyme forms finds its extremes in the eukaryal nucleus where RNA-based catalysis by complex ribonucleoproteins in some organisms contrasts with single-polypeptide enzymes in others. Such structural contrast suggests associated functional differences, and the complexity of the ribonucleoprotein was indeed proposed to broaden the enzyme's functionality beyond tRNA processing. To explore functional overlap and differences between most divergent forms of RNase P, we replaced the nuclear RNase P of Saccharomyces cerevisiae, a 10-subunit ribonucleoprotein, with Arabidopsis thaliana PRORP3, a single monomeric protein. Surprisingly, the RNase P-swapped yeast strains were viable, displayed essentially unimpaired growth under a wide variety of conditions, and, in a certain genetic background, their fitness even slightly exceeded that of the wild type. The molecular analysis of the RNase P-swapped strains showed a minor disturbance in tRNA metabolism, but did not point to any RNase P substrates or functions beyond that. Altogether, these results indicate the full functional exchangeability of the highly dissimilar enzymes. Our study thereby establishes the RNase P family, with its combination of structural diversity and functional uniformity, as an extreme case of convergent evolution. It moreover suggests that the apparently gratuitous complexity of some RNase P forms is the result of constructive neutral evolution rather than reflecting increased functional versatility.

  14. 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

    2012-03-02

    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.

  15. 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.

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

    Science.gov (United States)

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

    2018-01-01

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

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  19. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-01

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

  1. 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.

  2. 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.

  3. Highly water-soluble ruthenium(II terpyridine coordination compounds form stable adducts with blood-borne metal transporting proteins

    Directory of Open Access Journals (Sweden)

    Marija Nišavić

    2018-03-01

    Full Text Available Three coordination compounds of ruthenium(II, belonging to a recently synthesised series of water-soluble compounds of general formula mer-[Ru(L3(N-NCl]Cl, where L3 = 4'-chloro-2,2':6',2″-terpyridine (Cl-tpy, N-N = ethylenediamine (en, 1,2-diaminocyclohexane (dach or 2,2'-bipyridine (bpy, have shown strong binding to calf thymus DNA and moderate in vitro cytotoxicity towards cancer cell lines. Knowing that serum proteins play a crucial role in the transport and deactivation of ruthenium drugs, we have conducted a detailed study of their interactions with two major metal-transporting serum proteins, albumin and transferrin, and it is presented herein. Ruthenated protein adducts were formed with various concentrations of the three compounds and then separated from the unbound portions by ultrafiltration through 10 kDa cut-off centrifugal filter units. The stoichiometry of binding was determined using inductively coupled plasma optical emission spectrometry. One mol of albumin bound up to 7, 8.5 and 1.5 mol of compound 1 ([Ru(Cl-tpy(enCl][Cl], 2 ([Ru(Cl-tpy(dachCl][Cl] and 3 ([Ru(Cl-tpy(bpyCl][Cl], respectively. One mol of transferrin bound up to 3, 3.5 and 0.4 mol of 1, 2 and 3, respectively. The affinity of albumin and transferrin for the three ruthenium compounds was evaluated using fluorescence quenching. The binding constants for 1 and 2 lay within the range 104–105 M−1, suggesting moderate-to-strong attachment to albumin. Both compounds showed much lower affinity for transferrin (102–103 M−1. Compound 3 bound weakly to each studied protein. High resolution ESI qTOF mass spectra of albumin before and after binding of 1 revealed the high stoichiometry of binding. Although the binding of the compounds 1–3 to albumin and transferrin did not affect proteins’ secondary structure much, their tertiary structures underwent some alterations, as deduced from the circular dichroism study. Changes in the stability of albumin, after

  4. Analysis of molecular forms of urine Retinol-Binding Protein in Fanconi Syndrome and design of an accurate immunoassay.

    Science.gov (United States)

    Burling, Keith A; Cutillas, Pedro R; Church, David; Lapsley, Marta; Norden, Anthony G W

    2012-02-18

    Retinol-Binding Protein in urine (uRBP), a biomarker for the proximal renal tubular disease of congenital and acquired Fanconi Syndrome (FS) occurs in multiple forms. However these have not had quantitative mass spectrometric (MS) analysis, nor is there a validated assay for defined molecular species of uRBP with linearity on sample dilution. A 'Top-down' MS approach identified distinct forms of uRBP differing by only one amino acid. Based on this, we designed a dual-monoclonal antibody-based fluorescence immunoassay calibrated with intact plasma RBP4. LC-MS showed that uRBP in FS (one Dent disease urine) comprised intact plasma RBP4 and C-terminal-truncated RBP4, desL-RBP4 and desLL-RBP4 in molar ratio 2:2:1. DELFIA® assay calibrated with plasma RBP4, formulated with two monoclonal antibodies (HyTest, Finland), mAb48 for capture and biotinylated-mAb42 for detection, provided good sensitivity (1 μg/L), working range>500 μg/L and good linearity on sample dilution. The three predominant forms of uRBP were equipotent over the assay working range. uRBP reference range was <3 μg/mmol creatinine and FS patients had concentrations of 1000-5000 μg/mmol creatinine. Using 'Top-down' MS analysis of uRBP we devised an accurate, linear, fluorescence immunoassay with defined RBP molecular targets optimal for uRBP measurement. Discrimination of elevated uRBP from the upper limit of normal was some 10-fold greater than previous assays. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Low-cysteine alpha-keratins and corneous beta-proteins are initially formed in the regenerating tail epidermis of lizard.

    Science.gov (United States)

    Alibardi, L; Michieli, F; Dalla Valle, L

    2017-01-01

    During tail regeneration in lizards, the stratified regenerating epidermis progressively gives rise to neogenic scales that form a new epidermal generation. Initially, a soft, un-scaled, pliable, and extensible epidermis is formed that is progressively replaced by a resistant but non-extensible scaled epidermis. This suggests that the initial corneous proteins are later replaced with harder corneous proteins. Using PCR and immunocytochemistry, the present study shows an upregulation in the synthesis of low-cysteine type I and II alpha-keratins and of corneous beta-proteins with a medium cysteine content and a low content in glycine (formerly termed beta-keratins) produced at the beginning of epidermal regeneration. Quantitative PCR indicates upregulation in the production of alpha-keratin mRNAs, particularly of type I, between normal and the thicker regenerating epidermis. PCR-data also indicate a higher upregulation for cysteine-rich corneous beta-proteins and a high but less intense upregulation of low glycine corneous protein mRNAs at the beginning of scale regeneration. Immunolabeling confirms the localization of these proteins, and in particular of beta-proteins with a medium content in cysteine initially formed in the wound epidermis and later in the differentiating corneous layers of regenerating scales. It is concluded that the wound epidermis initially contains alpha-keratins and corneous beta-proteins with a lower cysteine content than more specialized beta-proteins later formed in the mature scales. These initial corneous proteins are likely related to the pliability of the wound epidermis while more specialized alpha-keratins and beta-proteins richer in glycine and cysteine are synthesized later in the mature and inflexible scales. J. Morphol. 278:119-130, 2017. ©© 2016 Wiley Periodicals,Inc. © 2016 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Novitskaya, V; Grigorian, M; Kriajevska, M

    2000-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  8. Identification of a phosphorylated non-structural form of the P protein of Newcastle disease virus and analysis of P multimers.

    Science.gov (United States)

    Hightower, L E; Collins, P L; Smith, G W

    1984-09-01

    Two phosphorylated and two non-phosphorylated variants of P protein isolated from Newcastle disease virions are known. Here, a fifth form of P was identified using two-dimensional polyacrylamide gel electrophoresis and peptide mapping. P form 5 was phosphorylated; however, unlike the four known variants of P, the new form was not a major protein in virions, which suggested an intracellular function. The subunit composition of four electrophoretically distinct, disulphide-linked multimers of P from virions was determined. Each homomultimer was composed of at least three molecules of a different one of the four virion-associated P variants.

  9. Inhibitory effects of nontoxic protein volvatoxin A1 on pore-forming cardiotoxic protein volvatoxin A2 by interaction with amphipathic alpha-helix.

    Science.gov (United States)

    Wu, Pei-Tzu; Lin, Su-Chang; Hsu, Chyong-Ing; Liaw, Yen-Chywan; Lin, Jung-Yaw

    2006-07-01

    Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea. Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2/volvatoxin A1 molar ratio of 2. In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activities of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic alpha-helices but it lacks a heparin-binding site. This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane. By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide competition assay and in conjunction with pull-down and co-pull-down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex. Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic alpha-helices, with two molecules of volvatoxin A2, each of which contains one amphipathic alpha-helix. Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy.

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

    International Nuclear Information System (INIS)

    Brandolin, Gerard

    1983-01-01

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

  12. Subcellular localization of a PhoE-LacZ fusion protein in E. coli by protease accessibility experiments reveals an inner-membrane-spanning form of the protein

    NARCIS (Netherlands)

    Tommassen, J.P.M.; Kroon, T. de

    1987-01-01

    Protease accessibility experiments were employed to localize a PhoE-LacZ hybrid protein, encompassing a large N-terminal fragment of the outer membrane PhoE protein of E. coli, fused to β-galactosidase, at the subcellular level. In previous studies, this protein was shown to co-fractionate with the

  13. 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.

  14. Dissociated GαGTP and Gβγ Subunits are the Major Activated Form of Heterotrimeric Gi/o Proteins

    Czech Academy of Sciences Publication Activity Database

    Bondar, Alexey; Lazar, Josef

    2014-01-01

    Roč. 289, č. 3 (2014), s. 1271-1281 ISSN 0021-9258 Institutional support: RVO:67179843 Keywords : heterotrimeric G proteins * heterotrimeric G proteins * adrenergic receptor * membrane proteins * microscopic imaging * plasma membrane * potassium channels * two-photon polarization microscopy Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  15. Helicobacter pylori CheZ(HP) and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor.

    Science.gov (United States)

    Lertsethtakarn, Paphavee; Howitt, Michael R; Castellon, Juan; Amieva, Manuel R; Ottemann, Karen M

    2015-09-01

    Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities. © 2015 John Wiley & Sons Ltd.

  16. 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.

  17. A chemical approach for site-specific identification of NMR signals from protein side-chain NH₃⁺ groups forming intermolecular ion pairs in protein-nucleic acid complexes.

    Science.gov (United States)

    Anderson, Kurtis M; Nguyen, Dan; Esadze, Alexandre; Zandrashvili, Levani; Gorenstein, David G; Iwahara, Junji

    2015-05-01

    Protein-nucleic acid interactions involve intermolecular ion pairs of protein side-chain and DNA or RNA phosphate groups. Using three protein-DNA complexes, we demonstrate that site-specific oxygen-to-sulfur substitution in phosphate groups allows for identification of NMR signals from the protein side-chain NH3 (+) groups forming the intermolecular ion pairs. A characteristic change in their (1)H and (15)N resonances upon this modification (i.e., substitution of phosphate to phosphorodithioate) can represent a signature of an intermolecular ion pair. Hydrogen-bond scalar coupling between protein side-chain (15)N and DNA phosphorodithiaote (31)P nuclei provides direct confirmation of the intermolecular ion pair. The same approach is likely applicable to protein-RNA complexes as well.

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  19. Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori.

    Science.gov (United States)

    Müller, Stephan A; Pernitzsch, Sandy R; Haange, Sven-Bastiaan; Uetz, Peter; von Bergen, Martin; Sharma, Cynthia M; Kalkhof, Stefan

    2015-08-03

    Helicobacter pylori (H. pylori) is a ε-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence. We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome. Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-α inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori. Our study shows that SILAC can be employed to study protein abundance changes in H. pylori. We have chosen to establish SILAC for H. pylori because it facilitates fractionation on both, protein and peptide level and thus enables deep proteome coverage. Furthermore, SILAC allows robust and highly accurate protein quantification. The manuscript includes a detailed description of the applied method, suggestions for further improvement as well as a practical application. The investigation of differences between the coccoid and infectious spiral morphology of H. pylori with SILAC revealed the regulation of proteins that are involved in host

  20. 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.

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

    Science.gov (United States)

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

    2009-01-01

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

  2. Diversity of the 32-kilodalton protein gene may form a basis for species determination of potentially pathogenic mycobacterial species.

    Science.gov (United States)

    Soini, H; Viljanen, M K

    1997-03-01

    In this study, partial gene sequences of the mycobacterial 32-kDa protein gene were determined by PCR-based sequencing. A total of 50 strains representing 18 potentially pathogenic mycobacterial species were studied. In 10 cases, all three strains of the species studied were identical, and intraspecies variability was found in 6 cases. Thus, the 32-kDa protein gene may be a good target for identification of mycobacteria by PCR-based sequencing.

  3. Structural Biology of DNA (6-4 Photoproducts Formed by Ultraviolet Radiation and Interactions with Their Binding Proteins

    Directory of Open Access Journals (Sweden)

    Hideshi Yokoyama

    2014-11-01

    Full Text Available Exposure to the ultraviolet component of sunlight causes DNA damage, which subsequently leads to mutations, cellular transformation, and cell death. DNA photoproducts with (6-4 pyrimidine-pyrimidone adducts are more mutagenic than cyclobutane pyrimidine dimers. These lesions must be repaired because of the high mutagenic potential of (6-4 photoproducts. We here reviewed the structures of (6-4 photoproducts, particularly the detailed structures of the (6-4 lesion and (6-4 lesion-containing double-stranded DNA. We also focused on interactions with their binding proteins such as antibody Fabs, (6-4 photolyase, and nucleotide excision repair protein. The (6-4 photoproducts that bound to these proteins had common structural features: The 5'-side thymine and 3'-side pyrimidone bases of the T(6-4T segment were in half-chair and planar conformations, respectively, and both bases were positioned nearly perpendicularly to each other. Interactions with binding proteins showed that the DNA helices flanking the T(6-4T segment were largely kinked, and the flipped-out T(6-4T segment was recognized by these proteins. These proteins had distinctive binding-site structures that were appropriate for their functions.

  4. Protein-electrophoretic variations detected in the fat body of the solitary form of the desert locust Schistocerca gregaria (Forskal)(Orthoptera: Acrididae)

    International Nuclear Information System (INIS)

    Aly, G.M.; Megahed, F.M.; Abd-Elsamie, E.M.

    2010-01-01

    The polyacrylamide gel electrophoresis (PAGE) was performed on the fat body tissues of the solitary form of the 5th nymph instar as well as three different phases of adult males and females namely newly emerged, yellow and non-yellow males (mature and nonmature), before oviposition and after oviposition females of Schistocerca gregaria. The total number of protein bands detected in the fat body of the 5th nymph instar amounted to 25 bands. Meanwhile, those detected in the fat body of newly emerged males, non-mature males and mature males were 22, 19, and 21 respectively. On the other hand, the numbers of protein bands detected in the newly emerged females were 28 bands. These bands varied in the successive female phases to be 21 and 30 bands before oviposition and after oviposition respectively. One protein band was detected in males only and considered as a male specific protein ( M-sp.) whereas, four bands were detected in females only and considered as female specific proteins ( F-sp.). Ten protein bands were considered as major fat body protein bands since they were commonly detected in all the investigated phases.

  5. Heat Shock Protein member A2 forms a stable complex with angiotensin converting enzyme and protein disulfide isomerase A6 in human spermatozoa.

    Science.gov (United States)

    Bromfield, Elizabeth G; McLaughlin, Eileen A; Aitken, Robert John; Nixon, Brett

    2016-02-01

    Given the importance of the chaperone Heat Shock Protein A2 (HSPA2) in the regulation of male fertility, this study aimed to identify and characterize additional proteins that may rely on the activity of this chaperone in human spermatozoa. In view of the findings in this study we propose that angiotensin converting enzyme (ACE) and protein disulfide isomerase A6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade. The molecular chaperone HSPA2 plays a pivotal role in the remodelling of the sperm surface during capacitation. Indeed, human spermatozoa that are deficient in HSPA2 protein expression lack the ability to recognize human oocytes, resulting in repeated IVF failure in a clinical setting. Moreover, our recent work has shown that defective HSPA2 function induced by oxidative stress leads to the aberrant surface expression of one of its interacting proteins, arylsulfatase A, and thus contributes to a loss of sperm-zona pellucida adhesion. Human spermatozoa were collected from fertile donors, capacitated and prepared for Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis. Protein complexes resolved via BN-PAGE were excised and their constituents were identified using mass spectrometry. The interactions between ACE, PDIA6 and HSPA2 were then confirmed using immunoprecipitation and proximity ligation assays and the localization of these proteins was assessed in isolated spermatozoa and commercially available human testis tissue sections. Finally, pharmacological inhibition of ACE was performed to assess the role of ACE in human sperm capacitation. Herein we have identified ACE and PDIA6 as potential HSPA2-interacting proteins and shown that this assemblage resides in membrane raft microdomains located in the peri-acrosomal region of the sperm head. Additionally, the surface expression of PDIA6, but not ACE, was shown to be dynamically regulated during sperm

  6. 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.

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

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-01

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

  10. Tor-Sch9 deficiency activates catabolism of the ketone body-like acetic acid to promote trehalose accumulation and longevity.

    Science.gov (United States)

    Hu, Jia; Wei, Min; Mirzaei, Hamed; Madia, Federica; Mirisola, Mario; Amparo, Camille; Chagoury, Shawna; Kennedy, Brian; Longo, Valter D

    2014-06-01

    In mammals, extended periods of fasting leads to the accumulation of blood ketone bodies including acetoacetate. Here we show that similar to the conversion of leucine to acetoacetate in fasting mammals, starvation conditions induced ketone body-like acetic acid generation from leucine in S. cerevisiae. Whereas wild-type and ras2Δ cells accumulated acetic acid, long-lived tor1Δ and sch9Δ mutants rapidly depleted it through a mitochondrial acetate CoA transferase-dependent mechanism, which was essential for lifespan extension. The sch9Δ-dependent utilization of acetic acid also required coenzyme Q biosynthetic genes and promoted the accumulation of intracellular trehalose. These results indicate that Tor-Sch9 deficiency extends longevity by switching cells to an alternative metabolic mode, in which acetic acid can be utilized for the storage of stress resistance carbon sources. These effects are reminiscent of those described for ketone bodies in fasting mammals and raise the possibility that the lifespan extension caused by Tor-S6K inhibition may also involve analogous metabolic changes in higher eukaryotes. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  11. Prevalence of Howell-Jolly body-like inclusions in HIV patients and their correlation with CD4 counts and HIV RNA viral load.

    Science.gov (United States)

    Chang, Brian; Huang, Richard Sheng Poe; Dasgupta, Amitava; Nguyen, Nghia; Wahed, Amer

    2015-01-01

    Previous reports have described the rare occurrence of detached nuclear fragments resembling Howell-Jolly bodies within neutrophils from HIV patients, organ-transplant recipients, and patients on immunosuppressive drugs. To date, their potential clinical significance is unknown, and pathologists tend to disregard their presence. Our study sought to find a correlation between these inclusions and the overall disease state, specifically within the HIV patient population. Eighty-three peripheral smears, all from different patients, were examined for the presence of inclusions and compared with recent CD4 counts and HIV RNA viral loads. Six cases contained inclusions, yielding a prevalence of 7.2%. These six patients had a mean CD4 count of 546±305 cells/μL compared to 247±242 cells/μL in those lacking inclusions (pHowell-Jolly body-like inclusions may be viewed as a potential biomarker indicative of a low risk for disease progression and/or good response to therapy based upon higher CD4 counts and relatively favorable viral loads. © 2015 by the Association of Clinical Scientists, Inc.

  12. Tor-Sch9 deficiency activates catabolism of the ketone body-like acetic acid to promote trehalose accumulation and longevity

    Science.gov (United States)

    Hu, Jia; Wei, Min; Mirzaei, Hamed; Madia, Federica; Amparo, Mario; Chagoury, Shawna; Kennedy, Brian; Longo, Valter D

    2014-01-01

    In mammals, extended periods of fasting leads to the accumulation of blood ketone bodies including acetoacetate. Here we show that similar to the conversion of leucine to acetoacetate in fasting mammals, starvation conditions induced ketone body-like acetic acid generation from leucine in S. cerevisiae. Whereas wild-type and ras2Δ cells accumulated acetic acid, long-lived tor1Δ and sch9Δ mutants rapidly depleted it through a mitochondrial acetate CoA transferase-dependent mechanism, which was essential for lifespan extension. The sch9Δ-dependent utilization of acetic acid also required coenzyme Q biosynthetic genes and promoted the accumulation of intracellular trehalose. These results indicate that Tor-Sch9 deficiency extends longevity by switching cells to an alternative metabolic mode, in which acetic acid can be utilized for the storage of stress resistance carbon sources. These effects are reminiscent of those described for ketone bodies in fasting mammals and raise the possibility that the lifespan extension caused by Tor-S6K inhibition may also involve analogous metabolic changes in higher eukaryotes. PMID:24649827

  13. 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

    immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular......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...... domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system....

  14. 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.

  15. 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.

  16. Genomic distribution of the small multidrug resistance protein EmrE over 29 Escherichia coli strains reveals two forms of the protein

    NARCIS (Netherlands)

    Kolbusz, Magdalena A.; Slotboom, Dirk J.; Lolkema, Juke S.

    Analysis of the genomes of 29 Escherichia coli strains revealed two different versions of the EmrE protein, a member of the small multidrug resistance family. The versions are different in length and contain 110 residues (EMRE110) and 165 residues (EMRE165). The N-terminal extension found in the

  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

  18. 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.

  19. The Yersinia enterocolitica phage shock proteins B and C can form homodimers and heterodimers in vivo with the possibility of close association between multiple domains.

    Science.gov (United States)

    Gueguen, Erwan; Flores-Kim, Josué; Darwin, Andrew J

    2011-10-01

    The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.

  20. The Yersinia enterocolitica Phage Shock Proteins B and C Can Form Homodimers and Heterodimers In Vivo with the Possibility of Close Association between Multiple Domains▿ †

    Science.gov (United States)

    Gueguen, Erwan; Flores-Kim, Josué; Darwin, Andrew J.

    2011-01-01

    The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces. PMID:21856846

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

    Science.gov (United States)

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

    2013-01-01

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

  2. 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.

  3. PseAAC-General: Fast Building Various Modes of General Form of Chou’s Pseudo-Amino Acid Composition for Large-Scale Protein Datasets

    Directory of Open Access Journals (Sweden)

    Pufeng Du

    2014-02-01

    Full Text Available The general form pseudo-amino acid composition (PseAAC has been widely used to represent protein sequences in predicting protein structural and functional attributes. We developed the program PseAAC-General to generate various different modes of Chou’s general PseAAC, such as the gene ontology mode, the functional domain mode, and the sequential evolution mode. This program allows the users to define their own desired modes. In every mode, 544 physicochemical properties of the amino acids are available for choosing. The computing efficiency is at least 100 times that of existing programs, which makes it able to facilitate the extensive studies on proteins and peptides. The PseAAC-General is freely available via SourceForge. It runs on both Linux and Windows.

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

    Science.gov (United States)

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

    2016-10-28

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

  5. A fusion between domains of the human bone morphogenetic protein-2 and maize 27 kD gamma-zein accumulates to high levels in the endoplasmic reticulum without forming protein bodies in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Valentina eCeresoli

    2016-03-01

    Full Text Available Human Bone Morphogenetic Protein-2 (hBMP2 is an osteoinductive agent physiologically involved in bone remodelling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD -zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

  6. Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

    Science.gov (United States)

    Roberts, Gareth A; Chen, Kai; Bower, Edward K M; Madrzak, Julia; Woods, Arcadia; Barker, Amy M; Cooper, Laurie P; White, John H; Blakely, Garry W; Manfield, Iain; Dryden, David T F

    2013-01-01

    ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res37, 4887–4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction. Structured digital abstract ArdA and ArdA bind by molecular sieving (1, 2) ArdA and ArdA bind by cosedimentation in solution (1, 2) PMID:23910724

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2012-12-01

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

  9. 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.

  10. Application of atomic force microscopy to protein anatomy:. Imaging of supramolecular structures of self-assemblies formed from synthetic peptides

    Science.gov (United States)

    Shibata-Seki, T.; Masai, J.; Ogawa, Y.; Sato, K.; Yanagawa, H.

    This paper reports morphological studies of structures of self-assemblies from synthetic peptide fragments with the use of atomic force microscope (AFM) and transmission electron microscope (TEM). Two systems of synthetic peptides have been examined: one is peptides from barnase (a ribonuclease) and the other is those from tau protein (Alzheimer's disease-related protein). The AFM observation was carried out by using a commercially available AFM operated in the tapping mode in air. The general appearance in shape and size of the peptide assemblies in AFM images was essentially similar to that in TEM images, except that the AFM images provide us with fruitful three-dimensional information about the assemblies. For assemblies from barnase peptides, possible formation processes of the supramolecular structures from the corresponding peptide fragment have been proposed on the basis of the AFM images.

  11. Effect of ice storage on the functional properties of proteins from a few species of fresh water fish (Indian major carps) with special emphasis on gel forming ability.

    Science.gov (United States)

    Mehta, Naresh Kumar; Elavarasan, K; Reddy, A Manjunatha; Shamasundar, B A

    2014-04-01

    In the present study the effect of ice storage on physico-chemical and functional properties of proteins from Indian major carps with special emphasis on gel forming ability have been assessed for a period of 22 days. The solubility profile of proteins in high ionic strength buffer and calcium adenosine triphosphatase (ATPase) enzyme activity reduced significantly (p ice storage. The major protein fraction showed association-dissociation-denaturation phenomenon during ice storage as revealed by gel filtration profile and viscosity measurements. The gel forming ability of three fish species both in fresh and during different periods of ice storage was assessed by measuring the gel strength of heat induced gel. Among the three species the gel strength of the gel obtained from Catla catla and Cirrhinus mrigala was higher (586 and 561 g.cm) than the gel obtained from Labeo rohita (395 g.cm) in fresh condition. The gel forming ability of three species was significantly affected (p ice storage. The TVB-N values of fish meat as a function of ice storage was within the prescribed limit up to 17 days of the ice storage.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2004-01-01

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

  14. Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and α2-macroglobulin

    International Nuclear Information System (INIS)

    Chhatwal, G.S.; Albohn, G.; Blobel, H.

    1987-01-01

    Binding of 125 I-labeled α 2 -macroglobulin (α 2 M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native α 2 M, and those of group C reacted only with α 2 M-trypsin complex. Binding of α 2 M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 x 10 -7 M, and the number of binding sites was calculated to be 18,000 per streptococcus. The α 2 M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native α 2 M in Western blots (immunoblots). Interaction of purified binding protein with α 2 M led to a change in the conformation of α 2 M similar to that obtained by α 2 M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of α 2 M is thus a novel feature of α 2 M reactivity

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

    Science.gov (United States)

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

    2016-11-01

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

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

    Science.gov (United States)

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

    2017-10-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  18. Effects of Physical Form and Urea Treatment of Rice Straw on Rumen Fermentation, Microbial Protein Synthesis and Nutrient Digestibility in Dairy Steers

    Directory of Open Access Journals (Sweden)

    P. Gunun

    2013-12-01

    Full Text Available This study was designed to determine the effect of physical form and urea treatment of rice straw on rumen fermentation, microbial protein synthesis and nutrient digestibility. Four rumen-fistulated dairy steers were randomly assigned according to a 2 (2 factorial arrangement in a 4 (4 Latin square design to receive four dietary treatments. Factor A was roughage source: untreated rice straw (RS and urea-treated (3% rice straw (UTRS, and factor B was type of physical form of rice straw: long form rice straw (LFR and chopped (4 cm rice straw (CHR. The steers were offered the concentrate at 0.5% body weight (BW /d and rice straw was fed ad libitum. DM intake and nutrient digestibility were increased (p0.05, except propionic acid (C3 was increased (p<0.05 in UTRS fed group. Nitrogen (N balance was affected by urea treatment (p<0.05. Microbial protein synthesis (MCP synthesis were greater by UTRS and CHR group (p<0.05. The efficiency of microbial N synthesis was greater for UTRS than for RS (p<0.05. From these results, it can be concluded that using the long form combined with urea treatment of rice straw improved feed intake, digestibility, rumen fermentation and efficiency of microbial N synthesis in crossbred dairy steers.

  19. The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release.

    Directory of Open Access Journals (Sweden)

    Smita Raghava

    2011-06-01

    Full Text Available Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.

  20. Combination of collagen and fibronectin to design biomimetic interfaces: Do these proteins form layer-by-layer assemblies?

    Science.gov (United States)

    Mauquoy, Sara; Dupont-Gillain, Christine

    2016-11-01

    Layer-by-layer (LbL) assembly is a surface modification method which may bring complexity to biointerfaces designed to control cell-material interactions. This work aims at investigating the LbL assembly of two extracellular matrix proteins, collagen (Col) and fibronectin (Fn), on polystyrene substrates. LbL assembly, which is widely applied to polyelectrolytes, is not easily transferred to proteins. Different buffers and conditions are tested, and LbL assembly is compared to the simultaneous adsorption of Fn and Col. Build-up and properties of the films are monitored using quartz crystal microbalance, ellipsometry, water contact angle measurements, and atomic force microscopy. Results show that denatured Col leads to smoother films, and that the addition of a polyethyleneimine anchoring layer increases film thickness. A more regular construction and thicker films are obtained with Hepes (pH 7.4) compared to other buffers. However, the LbL assembly is not sustainable and stops after the deposition of a few layers. Films obtained by simultaneous adsorption have lower water contact angles, different morphologies, lower water content and are as thick or thicker compared to the ones prepared by the LbL method. The present work shows that collagen and fibronectin are not involved in a true LbL assembly process. The obtained biointerfaces however exhibit different properties compared to those obtained by the one-step adsorption of these proteins. These differences could be exploited to control cell fate. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    Science.gov (United States)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  2. Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

    Science.gov (United States)

    Hwang, T C; Horie, M; Gadsby, D C

    1993-05-01

    The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some

  3. The effects of cell phone electromagnetic fields on the behavior of single OmpF nanochannel forming protein: a practical approach

    Directory of Open Access Journals (Sweden)

    Mohammadzadeh M

    2013-05-01

    Full Text Available Background: Widespread of telecommunication systems in recent years, have raised the concerns on the possible danger of cell phone radiations on human body. Thus, the study of the electromagnetic fields on proteins, particularly the membrane nano channel forming proteins is of great importance. These proteins are responsible for keeping certain physic-chemical condition within cells and managing cell communication. Here, the effects of cell phones radiation on the activity of a single nanopore ion channel forming protein, OmpF, have been studied biophysically.Methods: Planar lipid bilayers were made based on Montal and Muller technique, and the activity of single OmpF channel reconstituted by electrical shock was recorded and analyzed by means of voltage-clamp technique at 20 ˚C. The planar lipid bilayers were formed from the monolayers made on a 60 μm diameter aperture in the 20 μm thick Teflon film that separated two (cis and trans compartments of the glass chamber. In this practical approach we were able to analyze characteristics of an individual channel at different chemical and physical experimental conditions. The voltage clamp was used to measure the channel’s conductance, voltage sensitivity, gating patterns in time scales as low as microseconds in real time.  Results: Our results showed that exposure of single voltage dependent channel, OmpF, to EMF of cell phone at high-frequency has a significant influence on the voltage sensitivity, gating properties and substate numbers of the single channel but has no effect on single-channel conductance. Regarding to the relaxation time, the channel also recovers in the millisecond time range when the field is removed. Conclusion: We observed an increase in the voltage sensitivity of the OmpF single channel while it had no effect on the single-channel conductance, which is remained to be further elucidated.

  4. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    Science.gov (United States)

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

  5. Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein.

    OpenAIRE

    Burns, J L; Clark, D K

    1992-01-01

    The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibio...

  6. 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...... and concepts from real designs by studying form in abstract contexts. The challenge for the first approach is how to support students in decoupling form from the work as a whole. The challenge for the second approach is how to translate general form into real design. Hence, choosing between the two approaches...

  7. Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

    Directory of Open Access Journals (Sweden)

    Christine Pampeno

    Full Text Available The laminin receptor (LamR is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C and PrP(Sc. Indeed, LamR is a receptor for PrP(C. Whether LamR interacts with PrP(Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

  8. Variation in the coding and 3' untranslated regions of the porcine prolactin receptor short form modifies protein expression and function.

    Science.gov (United States)

    Trott, Josephine F; Freking, Bradley A; Hovey, Russell C

    2014-02-01

    The actions of prolactin (PRL) are mediated by both long (LF) and short isoforms (SF) of the PRL receptor (PRLR). Here, we report on a genetic and functional analysis of the porcine PRLR (pPRLR) SF. Three single nucleotide polymorphisms (SNPs) within exon 11 of the pPRLR-SF give rise to four amino acid haplotypes of the intracellular domain. We identified the dimorphic insertion of a short interspersed repetitive DNA element (PRE-1) along with 32 SNPs and four other insertion/deletion sites within the 3' untranslated region (UTR) of pPRLR-SF. The PRE-1 element reduced protein translation in vitro by 75%, whereas the combination of 10 SNPs and one insertion/deletion decreased translation by 50%. Full-length cDNAs for all four haplotypes of pPRLR-SF were cloned behind the elongation factor 1-alpha promoter and functionally analyzed in vitro. None of the haplotypes could initiate transcription from the ß-casein promoter, whereas all four were dominant negatives against PRL-activation of the pPRLR-LF. Two of the haplotypes completely inhibited pPRLR-LF activity at a four-fold excess, whereas the others required a six-fold excess to impart the same effect. The ligand binding affinities of the pPRLR-SF haplotypes did not differ. Expression of the pPRLR-SF increased linearly during gestation in the endometrium and was hormonally regulated in a tissue-specific manner in the mammary glands and uterus. In conclusion, we identified a PRE-1 and other SNPs in the pPRLR-SF 3' UTR that reduce protein expression and four haplotypes of the pPRLR-SF that suppress pPRLR-LF signaling and may differentially impact the phenotypic effects of PRL in vivo. © 2013 Stichting International Foundation for Animal Genetics.

  9. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    Directory of Open Access Journals (Sweden)

    Annarita Fiorillo

    Full Text Available The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3, unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  10. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    KAUST Repository

    Fiorillo, Annarita

    2014-03-21

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  11. Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

    Science.gov (United States)

    Salari, Farhad; Vahedi, Fatemeh; Chamani, Jamshidkhan; Varasteh, Abdolreza; Ketabdar, Hanieh; Sankian, Mojtaba

    2015-01-01

    Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  12. Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

    Science.gov (United States)

    Awayn, N H; Rosenberg, M F; Kamis, A B; Aleksandrov, L A; Riordan, J R; Ford, R C

    2005-11-01

    Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

  13. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

    Directory of Open Access Journals (Sweden)

    Sébastien Besteiro

    2009-02-01

    Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

  14. Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

    Science.gov (United States)

    Lattanzi, W; Parrilla, C; Fetoni, A; Logroscino, G; Straface, G; Pecorini, G; Stigliano, E; Tampieri, A; Bedini, R; Pecci, R; Michetti, F; Gambotto, A; Robbins, P D; Pola, E

    2008-10-01

    Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DmuCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

  15. The Holin Protein of Bacteriophage PRD1 Forms a Pore for Small-Molecule and Endolysin Translocation

    Science.gov (United States)

    Žiedaitė, Gabija; Daugelavičius, Rimantas; Bamford, Jaana K. H.; Bamford, Dennis H.

    2005-01-01

    PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ∼5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids. PMID:16030234

  16. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

    DEFF Research Database (Denmark)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

    2012-01-01

    and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain....... Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress...

  17. Detection of AA76, a Common Form of Amyloid A Protein, as a Way of Diagnosing AA Amyloidosis.

    Science.gov (United States)

    Sato, Junji; Okuda, Yasuaki; Kuroda, Takeshi; Yamada, Toshiyuki

    2016-01-01

    Reactive amyloid deposits consist of amyloid A (AA) proteins, the degradation products of serum amyloid A (SAA). Since the most common species of AA is the amino terminal portion produced by cleavage between residues 76 and 77 of SAA (AA76), the presence of AA76 in tissues could be a consequence of AA amyloid deposition. This study assessed the diagnostic significance of the detection of AA76 for AA amyloidosis using two different approaches. Biopsy specimens (n=130 from 54 subjects) from gastroduodenal mucosa or abdominal fat (n=9 from 9 subjects) of patients who had already been diagnosed with or were suspected of having AA amyloidosis were used. Fixed mucosal sections were subjected to immunohistochemistry using a newly developed antibody recognizing the carboxyl terminal end of AA76 (anti-AA76). The non-fixed materials from gastroduodenal mucosa or abdominal fat were subjected to immunoblotting for detection of the size of AA76. Among the gastroduodenal specimens (n=115) from already diagnosed patients, the positive rates of Congo red staining, immunohistochemistry using anti-AA76, and immunoblotting were 68.4%, 73.0%, and 92.2%, respectively. The anti-AA76 did not stain the supposed SAA in the blood or leakage, which was stained by anti-SAA antibody. AA76 was not detected either by immunohistochemistry or by immunoblot in the materials from patients in whom AA amyloidosis had been ruled out. In the abdominal fat, the immunoblot detected AA76 in 8 materials from 8 already diagnosed patients and did not in 1 patient whose gastroduodenal mucosa was negative. In conclusion, the detection of AA76 may alter the ability to diagnose AA amyloidosis. In immunohistochemistry for fixed specimens, the new anti-AA76 antibody can improve the specificity. Immunoblot for non-fixed materials, which can considerably improve the sensitivity, should be beneficial for small materials like abdominal fat. © 2016 by the Association of Clinical Scientists, Inc.

  18. HLA-F complex without peptide binds to MHC class I protein in the open conformer form.

    Science.gov (United States)

    Goodridge, Jodie P; Burian, Aura; Lee, Ni; Geraghty, Daniel E

    2010-06-01

    HLA-F has low levels of polymorphism in humans and is highly conserved among primates, suggesting a conserved function in the immune response. In this study, we probed the structure of HLA-F on the surface of B lymphoblastoid cell lines and activated lymphocytes by direct measurement of peptide binding to native HLA-F. Our findings suggested that HLA-F is expressed independently of bound peptide, at least in regard to peptide complexity profiles similar to those of either HLA-E or classical MHC class I (MHC-I). As a further probe of native HLA-F structure, we used a number of complementary approaches to explore the interactions of HLA-F with other molecules, at the cell surface, intracellularly, and in direct physical biochemical measurements. This analysis demonstrated that HLA-F surface expression was coincident with MHC-I H chain (HC) expression and was downregulated upon perturbation of MHC-I HC structure. It was further possible to directly demonstrate that MHC-I would interact with HLA-F only when in the form of an open conformer free of peptide and not as a trimeric complex. This interaction was directly observed by coimmunoprecipitation and by surface plasmon resonance and indirectly on the surface of cells through coincident tetramer and MHC-I HC colocalization. These data suggest that HLA-F is expressed independently of peptide and that a physical interaction specific to MHC-I HC plays a role in the function of MHC-I HC expression in activated lymphocytes.

  19. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Adenosine-uridine-rich element is one of the required cis-elements for epimastigote form stage-specific gene expression of the congolense epimastigote specific protein.

    Science.gov (United States)

    Suganuma, Keisuke; Mochabo, Kennedy Miyoro; Hakimi, Hassan; Yamasaki, Shino; Yamagishi, Junya; Asada, Masahito; Kawazu, Shin-Ichiro; Inoue, Noboru

    2013-09-01

    It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3' untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3'UTR. In the present study, we identified the adenosine and uridine rich region in the cesp 3'UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3'UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and RBP complexes might specifically stabilize cesp mRNA in EMF. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. The shortest isoform of dystrophin (Dp40) interacts with a group of presynaptic proteins to form a presumptive novel complex in the mouse brain.

    Science.gov (United States)

    Tozawa, Takenori; Itoh, Kyoko; Yaoi, Takeshi; Tando, So; Umekage, Masafumi; Dai, Hongmei; Hosoi, Hajime; Fushiki, Shinji

    2012-04-01

    Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD.

  2. RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

    2017-02-01

    In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

  3. Ergothioneine prevents copper-induced oxidative damage to DNA and protein by forming a redox-inactive ergothioneine-copper complex.

    Science.gov (United States)

    Zhu, Ben-Zhan; Mao, Li; Fan, Rui-Mei; Zhu, Jun-Ge; Zhang, Ying-Nan; Wang, Jing; Kalyanaraman, Balaraman; Frei, Balz

    2011-01-14

    Ergothioneine (2-mercaptohistidine trimethylbetaine) is a naturally occurring amino acid analogue found in up to millimolar concentrations in several tissues and biological fluids. However, the biological functions of ergothioneine remain incompletely understood. In this study, we investigated the role of ergothioneine in copper-induced oxidative damage to DNA and protein, using two copper-containing systems: Cu(II) with ascorbate and Cu(II) with H(2)O(2) [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H(2)O(2)]. Oxidative damage to DNA and bovine serum albumin was measured as strand breakage and protein carbonyl formation, respectively. Ergothioneine (0.1-1.0 mM) provided strong, dose-dependent protection against oxidation of DNA and protein in both copper-containing systems. In contrast, only limited protection was observed with the purported hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Ergothioneine also significantly inhibited copper-catalyzed oxidation of ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous copper, but not cupric copper, as demonstrated by UV-visible and low-temperature electron spin resonance techniques. We conclude that ergothioneine is a potent, natural sulfur-containing antioxidant that prevents copper-dependent oxidative damage to biological macromolecules by forming a redox-inactive ergothioneine-copper complex.

  4. DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

    Directory of Open Access Journals (Sweden)

    Marcus Thein

    Full Text Available Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

  5. Chemical synthesis of a pore-forming antimicrobial protein, caenopore-5, by using native chemical ligation at a glu-cys site.

    Science.gov (United States)

    Medini, Karima; Harris, Paul W R; Hards, Kiel; Dingley, Andrew J; Cook, Gregory M; Brimble, Margaret A

    2015-01-19

    The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore-5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore-forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid-phase peptide synthesis and native chemical ligation. No γ-linked by-product was observed despite the use of a C-terminal Glu-thioester. The folding of the synthetic protein was confirmed by (1) H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore-5. The permeabilisation activities of the protein and of shortened analogues were evaluated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. High-resolution structures of Thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with NAD+ and in complex with NAD+ and triclosan

    International Nuclear Information System (INIS)

    Otero, José M.; Noël, Ann-Josée; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Wende, Wolfgang; Schierling, Benno; Pingoud, Alfred; Raaij, Mark J. van

    2012-01-01

    T. thermophilus enoyl-acyl carrier protein reductase was crystallized in the apo form, with NAD + bound and with NAD + and the inhibitor triclosan bound. The structures were solved by molecular replacement and refined at 1.50, 1.86 and 1.90 Å resolution, respectively. The structures are described, analysed and compared with those of enoyl-acyl carrier protein reductases from other species. Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD + and bound to NAD + plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional β-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR

  7. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  8. Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

    Science.gov (United States)

    Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

    2017-06-21

    Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

  9. Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein from Helicobacter pylori: Role of Multiple Metal Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rong; Munger, Christine; Asinas, Abdalin; Benoit, Stephane L.; Miller, Erica; Matte, Allan; Maier, Robert J.; Cygler, Miroslaw (McGill); (Georgia); (Biotech Res.)

    2010-10-22

    The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 {angstrom} resolution, bound to Cu{sup 2+} at 2.7 {angstrom} resolution, and bound to Ni{sup 2+} at 3.1 {angstrom} resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding. The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni{sup 2+} for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni{sup 2+}-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.

  10. PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.

    Science.gov (United States)

    Chevalier, Christophe; Al Bazzal, Ali; Vidic, Jasmina; Février, Vincent; Bourdieu, Christiane; Bouguyon, Edwige; Le Goffic, Ronan; Vautherot, Jean-François; Bernard, Julie; Moudjou, Mohammed; Noinville, Sylvie; Chich, Jean-François; Da Costa, Bruno; Rezaei, Human; Delmas, Bernard

    2010-04-23

    The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

  11. PB1-F2 Influenza A Virus Protein Adopts a β-Sheet Conformation and Forms Amyloid Fibers in Membrane Environments

    Science.gov (United States)

    Chevalier, Christophe; Al Bazzal, Ali; Vidic, Jasmina; Février, Vincent; Bourdieu, Christiane; Bouguyon, Edwige; Le Goffic, Ronan; Vautherot, Jean-François; Bernard, Julie; Moudjou, Mohammed; Noinville, Sylvie; Chich, Jean-François; Da Costa, Bruno; Rezaei, Human; Delmas, Bernard

    2010-01-01

    The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an α-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display β-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a β-sheet structure. Dynamic light scattering revealed that the presence of β-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation. PMID:20172856

  12. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

  13. Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.

    Science.gov (United States)

    Briegel, Ariane; Ortega, Davi R; Mann, Petra; Kjær, Andreas; Ringgaard, Simon; Jensen, Grant J

    2016-09-13

    Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

  14. Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein.

    Science.gov (United States)

    Wilson, Rona; Plinston, Chris; Hunter, Nora; Casalone, Cristina; Corona, Cristiano; Tagliavini, Fabrizio; Suardi, Silvia; Ruggerone, Margherita; Moda, Fabio; Graziano, Silvia; Sbriccoli, Marco; Cardone, Franco; Pocchiari, Maurizio; Ingrosso, Loredana; Baron, Thierry; Richt, Juergen; Andreoletti, Olivier; Simmons, Marion; Lockey, Richard; Manson, Jean C; Barron, Rona M

    2012-07-01

    The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

  15. Immediate-Early Protein ME53 Forms Foci and Colocalizes with GP64 and the Major Capsid Protein VP39 at the Cell Membranes of Autographa californica Multiple Nucleopolyhedrovirus-Infected Cells ▿ †

    Science.gov (United States)

    de Jong, Jondavid; Theilmann, David A.; Arif, Basil M.; Krell, Peter J.

    2011-01-01

    me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid. PMID:21775466

  16. Venezuelan equine Encephalitis virus capsid protein forms a tetrameric complex with CRM1 and importin alpha/beta that obstructs nuclear pore complex function.

    Science.gov (United States)

    Atasheva, Svetlana; Fish, Alexander; Fornerod, Maarten; Frolova, Elena I

    2010-05-01

    Development of the cellular antiviral response requires nuclear translocation of multiple transcription factors and activation of a wide variety of cellular genes. To counteract the antiviral response, several viruses have developed an efficient means of inhibiting nucleocytoplasmic traffic. In this study, we demonstrate that the pathogenic strain of Venezuelan equine encephalitis virus (VEEV) has developed a unique mechanism of nuclear import inhibition. Its capsid protein forms a tetrameric complex with the nuclear export receptor CRM1 and the nuclear import receptor importin alpha/beta. This unusual complex accumulates in the center channel of the nuclear pores and blocks nuclear import mediated by different karyopherins. The inhibitory function of VEEV capsid protein is determined by a short 39-amino-acid-long peptide that contains both nuclear import and supraphysiological nuclear export signals. Mutations in these signals or in the linker peptide attenuate or completely abolish capsid-specific inhibition of nuclear traffic. The less pathogenic VEEV strains contain a wide variety of mutations in this peptide that affect its inhibitory function in nuclear import. Thus, these mutations appear to be the determinants of this attenuated phenotype. This novel mechanism of inhibiting nuclear transport also shows that the nuclear pore complex is vulnerable to unusual cargo receptor complexes and sheds light on the importance of finely adjusted karyopherin-nucleoporin interactions for efficient cargo translocation.

  17. Dietary amino acids fed in free form and as protein components do not differently affect postprandial plasma insulin, glucagon, growth hormone and corticosterone responses in rats.

    Science.gov (United States)

    Nolles, J A; Van Straten, E M E; Bremer, B I; Koopmanschap, R E; Verstegen, M W A; Schreurs, V V A M

    2006-08-01

    This study examined, whether the postprandial fate of dietary amino acids from different amino acid sources is regulated by the responses of insulin, glucagon, corticosterone and growth hormone (GH). Male Wistar rats were cannulated in the vena jugularis and assigned to dietary groups. The diets contained 21% casein or the same amino acids in free form. In the free amino acid diets, methionine level was varied between the groups. The feed was supplied in two distinct meals. In previous experiments it was established that oxidative amino acid losses of the free amino acid diets and protein diets were different. After 3 weeks on those diets, it appeared that the differences in postprandial oxidative losses had been diminished. GH was measured every 12 min, from 144 min before the start of the experimental meal over the following 144 min. Insulin and corticosterone were measured six times from the start of the meal until 270 min after the meal. No differences have been observed between the hormonal responses to both meals at day 5 and at day 26. In conclusion, it has been found that the differences in the oxidative losses between protein and free amino acid meals are not mediated by the combined action of the insulin, glucagon, corticosterone and GH. Postprandial catabolism of amino acids is most probably regulated by substrate induction.

  18. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    DEFF Research Database (Denmark)

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  19. 1H, 15N, 13C Resonance Assignments of the Reduced and Active Form of Human Protein Tyrosine Phosphatase, PRL-1

    Science.gov (United States)

    Skinner, Andria L.; Laurence, Jennifer S.

    2009-01-01

    Phosphatase of regenerating liver-1 (PRL-1) is a novel target for potentially treating cancer metastases. Although its specific biochemical role in these processes has yet to be delineated, considerable evidence suggests the phosphatase activity of PRL-1 is required for promoting cancer and metastasis. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and functions using the CX5R consensus active site motif. Like other PTPases, PRL-1 is inhibited by oxidation at its active site Cys, however, disulfide bond formation occurs unusually readily in wild-type PRL-1. Chemical shift assignments are available for oxidized wild type, but numerous, substantial changes are observed in the spectra upon reduction. Because the reduced form is active, we sought to identify a stable mutant that would resist oxidation and be useful for facilitating drug screening and development using NMR-based assays. We present here NMR assignments for a full-length, reduced and active form of PRL-1, PRL-1-C170S-C171S, that is well suited for this purpose. PMID:19636948

  20. 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

    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 by...

  1. Analysis of p107-associated proteins: p107 associates with a form of E2F that differs from pRB-associated E2F-1

    DEFF Research Database (Denmark)

    Dyson, N; Dembski, M; Fattaey, A

    1993-01-01

    The binding of viral oncogenes to cellular proteins is thought to modulate the activities of these cellular targets. The p107 protein is targeted by many viral proteins, including adenovirus E1A, simian virus 40 large T antigen, and human papillomavirus type 16 E7 protein. A panel of monoclonal...

  2. The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

    Energy Technology Data Exchange (ETDEWEB)

    Makhov, A.M.; Simon, M.; Sen, A.; Yu, X.; Griffith, J. D.; Egelman, E. H.

    2009-02-20

    Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is {approx} 250 {angstrom}, with {approx} 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing {approx} 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

  3. An analysis of the levels of the soluble form of the endothelial protein C receptor in children with Henoch-Schönlein Purpura.

    Science.gov (United States)

    Cayci, Fatma Semsa; Ekim, Mesiha; Egin, Yonca; Gökce, Hafize; Yalcinkaya, Fatos; Ozcakar, Birsin; Akar, Nejat

    2015-03-01

    The pathogenesis of Henoch-Schönlein Purpura (HSP) has not been clearly defined. Inflammatory cytokines have been associated with HSP but there are only a few reports that have focused on coagulation. The endothelial protein C receptor (EPCR), which has anticoagulant and antiinflammatory activity, is the key component of the protein C pathway. Recent studies have implicated the soluble form of EPCR (sEPCR) in Wegener's granulomatosis, Behçet's disease, and systemic lupus erythematosus. The aim of this study was to evaluate the levels of sEPCR in HSP children. Twenty-two children with HSP and 17 healthy children were included. We found no significant differences (P > .05) between patient and control groups in the levels of von Willebrand factor and thrombomodulin. The median sEPCR values in the HSP group were lower than the control group (79 vs. 102 ng/mL, respectively) (P > .05). The mean sEPCR value in HSP patients with severe abdominal pain was lower than without (88.8 ± 54.9 vs. 108.2 ± 66.3 ng/mL, respectively) (P > .05). In addition, the mean IL-6 serum levels were significantly elevated in HSP patients during the acute stage of HSP (2.1 ± 1.7 vs. 1.5 ± 1.2 pg/mL, P = .038). We also observed a slight negative correlation between the levels of sEPCR and IL-6 (R = -.135, P > .05). To our knowledge, this was the first study to analyze sEPCR levels in HSP. Our results did not conclusively identify a direct role of sEPCR in HSP, but our findings warrant further investigations, especially in severe HSP cases characterized by gastrointestinal bleeding or renal involvement.

  4. Structure of an Odorant-Vinding Protein form the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive

    Energy Technology Data Exchange (ETDEWEB)

    N Leite; R Krogh; W Xu; Y Ishida; J Iulek; W Leal; G Oliva

    2011-12-31

    The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 {angstrom} resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six {alpha}-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this 'lid' may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

  5. Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro.

    Science.gov (United States)

    Chien, H H; Lin, W L; Cho, M I

    1999-05-01

    The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture

  6. Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

    International Nuclear Information System (INIS)

    Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

    2012-01-01

    Highlights: ► Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. ► Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. ► Only monomers of ANGPTL4 are present within THP-1 macrophages. ► Covalent oligomers of ANGPTL4 appear on cell surface and in medium. ► Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

  7. Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

    Energy Technology Data Exchange (ETDEWEB)

    Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Thulin, Petra; Ehrenborg, Ewa [Atherosclerosis Research Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm (Sweden); Olivecrona, Thomas [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Olivecrona, Gunilla, E-mail: Gunilla.Olivecrona@medbio.umu.se [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

  8. X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB.

    Science.gov (United States)

    Niemann, Hartmut H; Petoukhov, Maxim V; Härtlein, Michael; Moulin, Martine; Gherardi, Ermanno; Timmins, Peter; Heinz, Dirk W; Svergun, Dmitri I

    2008-03-21

    The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.

  9. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-11-01

    Full Text Available VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa, and the shorter VirF21 (21 kDa, lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.

  10. Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

    Science.gov (United States)

    Honda, Masayoshi; Okuno, Yusuke; Hengel, Sarah R.; Martín-López, Juana V.; Cook, Christopher P.; Amunugama, Ravindra; Soukup, Randal J.; Subramanyam, Shyamal; Fishel, Richard; Spies, Maria

    2014-01-01

    High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2–hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2–hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2–hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2–hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2–hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2–hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA. PMID:24395779

  11. Complex oligomeric structure of a truncated form of DdrA: A protein required for the extreme radio-tolerance of Deinococcus

    International Nuclear Information System (INIS)

    Gutsche, I.; Vujicic-Zagar, A.; Serre, L.; Siebert, X.; Servant, P.; Vannier, F.; Sommer, S.; Castaing, B.; Gallet, B.; Heulin, T.; De Groot, A.

    2008-01-01

    In order to preserve their genome integrity, organisms have developed elaborate tactics for genome protection and repair. The Deinococcus radiodurans bacteria famous for their extraordinary tolerance toward high doses of radiations or long period of desiccation, possess some specific genes with unknown function which are related to their survival in such extreme conditions. Among them, ddrA is an orphan gene specific of Deinococcus genomes. DdrA, the product of this gene was suggested to be a component of the DNA end protection system. Here we provide a three-dimensional reconstruction of the Deinococcus deserti DdrA(1-160) by electron microscopy. Although not functional in vivo, this truncated protein keeps its DNA binding ability at the wild-type level. DdrA(1-160) has a complex three-dimensional structure based on a hepta-meric ring that can self-associate to form a larger molecular weight assembly. We suggest that the complex architecture of DdrA plays a role in the substrate specificity and favors an efficient DNA repair. (authors)

  12. Bovine colostrum increases pore-forming claudin-2 protein expression but paradoxically not ion permeability possibly by a change of the intestinal cytokine milieu.

    Directory of Open Access Journals (Sweden)

    Peggy Bodammer

    Full Text Available An impaired intestinal barrier function is involved in the pathogenesis of inflammatory bowel disease (IBD. Several nutritional factors are supposed to be effective in IBD treatment but scientific data about the effects on the intestinal integrity remain scarce. Bovine colostrum was shown to exert beneficial effects in DSS-induced murine colitis, and the present study was undertaken to explore the underlying molecular mechanisms. Western blot revealed increased claudin-2 expression in the distal ileum of healthy mice after feeding with colostrum for 14 days, whereas other tight junction proteins (claudin-3, 4, 10, 15 remained unchanged. The colostrum-induced claudin-2 induction was confirmed in differentiated Caco-2 cells after culture with colostrum for 48 h. Paradoxically, the elevation of claudin-2, which forms a cation-selective pore, was neither accompanied by increased ion permeability nor impaired barrier function. In an in situ perfusion model, 1 h exposure of the colonic mucosa to colostrum induced significantly increased mRNA levels of barrier-strengthening cytokine transforming growth factor-β, while interleukine-2, interleukine-6, interleukine-10, interleukine-13, and tumor-necrosis factor-α remained unchanged. Thus, modulation of the intestinal transforming growth factor-β expression might have compensated the claudin-2 increase and contributed to the observed barrier strengthening effects of colostrum in vivo and in vitro.

  13. Influence of Go-like interactions on global shapes of energy landscapes in beta-barrel forming model proteins: inherent structure analysis and statistical temperature molecular dynamics simulation.

    Science.gov (United States)

    Kim, Jaegil; Keyes, Thomas

    2008-01-24

    Recently, the native-centric, coarse-grained "Go-like" protein model has gained a renewed popularity because of its computational simplicity and successful description of some key aspects of folding thermodynamics and kinetics. In the present paper, the properties of Go-like models are investigated in terms of the potential energy landscape (PEL). The non-native attractions of the beta-barrel forming BLN model 46-mer are scaled with a parameter 0 Go-like interactions brings about a significant modification in the occupation of low-lying IS by destabilizing the misfolding funnel into a higher energy region, strongly influencing the folding thermodynamics. The alteration of the PEL also induces a dramatic change in the folding mechanism, from a second-order-like collapse transition into a cooperative, first-order-like folding transition, occurring through a transient, intermediate state ensemble characterized by partially structured IS. The appearance of multiple van der Waals loops in the statistical temperature of the Go-like model is associated with the development of the intermediate states.

  14. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

    Science.gov (United States)

    Sarafanov, Andrey G; Makogonenko, Evgeny M; Pechik, Igor V; Radtke, Klaus-Peter; Khrenov, Alexey V; Ananyeva, Natalya M; Strickland, Dudley K; Saenko, Evgueni L

    2006-02-14

    Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

  15. El cuerpo como excusa: El diagnostico de la fibromialgia en una consulta de reumatología The body likes excuse: Diagnosing fibromyalgia in a rheumatologic clinic

    Directory of Open Access Journals (Sweden)

    Beatriz Tosal Herrero

    2008-03-01

    Full Text Available Introducción: El proceso de medicalización de la sociedad ha generado una dependencia de la biomedicina para entender, gestionar y legitimar la enfermedad. Como consecuencia surge una nueva forma de considerar los procesos corporales que transforma las identidades y subjetividades de los individuos y los grupos sociales. La enfermedad se concibe y se experimenta en el cuerpo en forma de signos y síntomas. Esta representación de la enfermedad compartida por expertos y enfermos durante el encuentro clínico se convierte en el lenguaje propio de la interacción. Esto ocurre incluso en casos, como el de la fibromialgia, en los que el diagnóstico se produce en ausencia de alteraciones ‘objetivas’ del cuerpo. Metodología: Observación participante, sobre la elaboración y comunicación del diagnóstico de fibromialgia en una consulta de reumatología. Resultados: Se analizan las representaciones sobre el cuerpo y la enfermedad que manejan tanto médicos como enfermos, y la utilización de los síntomas corporales como estrategia de legitimación e interacción durante el encuentro clínico. Conclusión: En la fibromialgia, a pesar de la ausencia de alteraciones biológicas ‘objetivas’, la relación sanitario-paciente se ve mediada por el cuerpo. Este es el lugar en el que se producen las intervenciones terapéuticas y el vehículo de la comunicación entre ambos.Introduction: The process of medicalization of the society has generated a dependence of the biomedicine to understand, to negotiate and to legitimate the illness. As consequence arises, a new form of considering the bodily processes that transforms the identities and the individuals' subjectivities. Persons conceive and experience the illness in the body as signs and symptoms. This representation of the illness shared by experts and patients during the clinical encounter it becomes the characteristic language of the interaction. This even happens in cases, as that of the

  16. Effects of ingesting protein with various forms of carbohydrate following resistance-exercise on substrate availability and markers of anabolism, catabolism, and immunity

    Directory of Open Access Journals (Sweden)

    Greenwood Michael

    2007-11-01

    Full Text Available Abstract Background Ingestion of carbohydrate (CHO and protein (PRO following intense exercise has been reported to increase insulin levels, optimize glycogen resynthesis, enhance PRO synthesis, and lessen the immuno-suppressive effects of intense exercise. Since different forms of CHO have varying glycemic effects, the purpose of this study was to determine whether the type of CHO ingested with PRO following resistance-exercise affects blood glucose availability and insulin levels, markers of anabolism and catabolism, and/or general immune markers. Methods 40 resistance-trained subjects performed a standardized resistance training workout and then ingested in a double blind and randomized manner 40 g of whey PRO with 120 g of sucrose (S, honey powder (H, or maltodextrin (M. A non-supplemented control group (C was also evaluated. Blood samples were collected prior to and following exercise as well as 30, 60, 90, and 120 min after ingestion of the supplements. Data were analyzed by repeated measures ANOVA or ANCOVA using baseline values as a covariate if necessary. Results Glucose concentration 30 min following ingestion showed the H group (7.12 ± 0.2 mmol/L to be greater than S (5.53 ± 0.6 mmol/L; p uIU/mL, H (150.1 ± 25.39 uIU/mL, and M (154.8 ± 18.9 uIU/mL were greater than C (8.7 ± 2.9 uIU/mL as was AUC with no significant differences observed among types of CHO. No significant group × time effects were observed among groups in testosterone, cortisol, the ratio of testosterone to cortisol, muscle and liver enzymes, or general markers of immunity. Conclusion CHO and PRO ingestion following exercise significantly influences glucose and insulin concentrations. Although some trends were observed suggesting that H maintained blood glucose levels to a better degree, no significant differences were observed among types of CHO ingested on insulin levels. These findings suggest that each of these forms of CHO can serve as effective sources of

  17. The Yeast Iron Regulatory Proteins Grx3/4 and Fra2 Form Heterodimeric Complexes Containing a [2Fe-2S] Cluster with Cysteinyl and Histidyl Ligation

    Energy Technology Data Exchange (ETDEWEB)

    Li, H.; Mapolelo, D; Dingra, N; Naik, S; Lees, N; Hoffman, B; Riggs-Gelasco, P; Huynh, B; Johnson, M; Outten, C

    2009-01-01

    The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S]{sup 2+} cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV?visible absorption and CD, resonance Raman, EPR, ENDOR, M{umlt o}ssbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.

  18. TprC/D (Tp0117/131), a Trimeric, Pore-Forming Rare Outer Membrane Protein of Treponema pallidum, Has a Bipartite Domain Structure

    Science.gov (United States)

    Anand, Arvind; Luthra, Amit; Dunham-Ems, Star; Caimano, Melissa J.; Karanian, Carson; LeDoyt, Morgan; Cruz, Adriana R.; Salazar, Juan C.

    2012-01-01

    Identification of Treponema pallidum rare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178–5194, 2010). TP0117/TP0131 (TprC/D), a member of the T. pallidum repeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprCFl) forms a β-barrel capable of integrating into lipid bilayers. Moreover, TprCFl increased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that in T. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog of Escherichia coli OmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprCN), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal β-barrel (TprCC). Syphilitic rabbits generate antibodies exclusively against TprCC, while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host. PMID:22389487

  19. Isolation of recombinant proteins from culture broth by co-precipitation with an amino acid carrier to form stable dry powders.

    Science.gov (United States)

    Moore, Barry D; Deere, Joseph; Edrada-Ebel, RuAngelie; Ingram, Andrew; van der Walle, Christopher F

    2010-08-01

    Protein-coated microcrystals can be generated by co-precipitation of protein and a water-soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co-precipitation with a L-glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co-elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co-precipitation process. Scale-up of the process was readily achieved through in-line mixing and subsequent reconstitution of the dried protein-coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L-glutamine facilitated further purification of the recombinant protein immobilized on the L-glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing. (c) 2010 Wiley Periodicals, Inc.

  20. Cluster analysis of historical and modern hard red spring wheat cultivars based on parentage and HPLC analysis of gluten forming proteins

    Science.gov (United States)

    In this study, 30 hard red spring (HRS) wheat cultivars released between 1910 and 2013 were analyzed to determine how they cluster in terms of parentage and protein data, analyzed by reverse-phase HPLC (RP-HPLC) of gliadins, and size-exclusion HPLC (SE-HPLC) of unreduced proteins. Dwarfing genes in...

  1. In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to S100 protein in released-active form

    Directory of Open Access Journals (Sweden)

    Gorbunov EA

    2015-11-01

    Full Text Available Evgeniy A Gorbunov, Irina A Ertuzun, Evgeniya V Kachaeva, Sergey A Tarasov, Oleg I EpsteinOOO “NPF “MATERIA MEDICA HOLDING”, Moscow, Russian FederationAbstract: Experimentally and clinically, it was shown that released-active form of antibodies to S100 protein (RAF of Abs to S100 exerts a wide range of pharmacological activities: anxiolytic, antiasthenic, antiaggressive, stress-protective, antihypoxic, antiischemic, neuroprotective, and nootropic. The purpose of this study was to determine the influence of RAF of Abs to S100 on major neurotransmitter systems (serotoninergic, GABAergic, dopaminergic, and on sigma receptors as well which are possibly involved in its mechanism of pharmacological activity. Radioligand binding assays were used for assessment of the drug influence on ligand–receptor interaction. [35S]GTPγS binding assay, cyclic adenosine monophosphate HTRF™, cellular dielectric spectroscopy assays, and assays based on measurement of intracellular concentration of Ca2+ ions were used for assessment of agonist or antagonist properties of the drug toward receptors. RAF of Abs to S100 increased radioligand binding to 5-HT1F, 5-HT2B, 5-HT2Cedited, 5-HT3, and to D3 receptors by 142.0%, 131.9%, 149.3%, 120.7%, and 126.3%, respectively. Also, the drug significantly inhibited specific binding of radioligands to GABAB1A/B2 receptors by 25.8%, and to both native and recombinant human sigma1 receptors by 75.3% and 40.32%, respectively. In the functional assays, it was shown that the drug exerted antagonism at 5-HT1B, D3, and GABAB1A/B2 receptors inhibiting agonist-induced responses by 23.24%, 32.76%, and 30.2%, respectively. On the contrary, the drug exerted an agonist effect at 5-HT1A receptors enhancing receptor functional activity by 28.0%. The pharmacological profiling of RAF of Abs to S100 among 27 receptor provides evidence for drug-related modification of major neurotransmitter systems.Keywords: dopamine agent, released

  2. Phosphorylation of a 72-kDa protein in PDGF-stimulated cells which forms complex with c-Crk, c-Fyn and Eps15

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Claesson-Welsh, L

    1997-01-01

    Ligand-induced activation of the beta-receptor for platelet-derived growth factor (PDGF) induces tyrosine phosphorylation of a number of downstream signaling proteins. In the present study, we used two-dimensional gel electrophoresis to characterize the spectrum of proteins phosphorylated...... in response to PDGF stimulation in porcine aortic endothelial cells expressing PDGF beta-receptors. Several previously known substrates for the PDGF beta-receptor were identified as well as a novel substrate of 72 kDa. The 72-kDa component could be co-immunoprecipitated in complex with the adaptor protein c...

  3. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  4. Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1

    Directory of Open Access Journals (Sweden)

    Yu Jialin

    2011-01-01

    Full Text Available Abstract Background Rice black-streaked dwarf virus (RBSDV, a member of the genus Fijivirus within the family Reoviridae, can infect several graminaceous plant species including rice, maize and wheat, and is transmitted by planthoppers. Although several RBSDV proteins have been studied in detail, functions of the nonstructural protein P6 are still largely unknown. Results In the current study, we employed yeast two-hybrid assays, bimolecular fluorescence complementation and subcellular localization experiments to show that P6 can self-interact to form punctate, cytoplasmic viroplasm-like structures (VLS when expressed alone in plant cells. The region from residues 395 to 659 is necessary for P6 self-interaction, whereas two polypeptides (residues 580-620 and 615-655 are involved in the subcellular localization of P6. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 395-659 region is also important for the P6-P9-1 interaction, and deleting any region of P9-1 abolishes this heterologous interaction. Conclusions RBSDV P6 protein has an intrinsic ability to self-interact and forms VLS without other RBSDV proteins or RNAs. P6 recruits P9-1 to VLS by direct protein-protein interaction. This is the first report on the functionality of RBSDV P6 protein. P6 may be involved in the process of viroplasm nucleation and virus morphogenesis.

  5. JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

    Science.gov (United States)

    Yin, T; Tsang, M L; Yang, Y C

    1994-10-28

    Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

  6. Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

    International Nuclear Information System (INIS)

    Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

    1987-01-01

    A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a λ gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source

  7. Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

    1987-12-01

    A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.

  8. Tomato leaf curl Kerala virus (ToLCKeV AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1

    Directory of Open Access Journals (Sweden)

    Mukherjee Sunil K

    2010-06-01

    Full Text Available Abstract Background Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study. Results AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro. Conclusions Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

  9. Thaumatin-like proteins and chitinases, the haze-forming proteins of wine, accumulate during ripening of grape (Vitis vinifera) berries and drought stress does not affect the final levels per berry at maturity.

    Science.gov (United States)

    Pocock, K F; Hayasaka, Y; McCarthy, M G; Waters, E J

    2000-05-01

    Thaumatin-like proteins and chitinases, which are pathogenesis-related (PR) proteins, were the major soluble protein components of grapes from five cultivars of Vitis vinifera. This dominance of PR proteins was apparent at berry softening (véraison) and then throughout berry development for the Muscat of Alexandria, Sultana, and Shiraz cultivars and in the berries of the Sauvignon Blanc and Pinot Noir cultivars examined at commercial maturity. The M(r) of the major thaumatin-like protein from Muscat of Alexandria grapes was 21 272, and those of the three major chitinases from this cultivar, ChitB, ChitC, and ChitD, were 25 588, 25 410, and 25 457, respectively. The vines in the study were irrigated and showed no obvious signs of disease. Shiraz vines that had not been irrigated throughout the season were clearly water stressed, but had levels of PR proteins in the berry similar to vines that had been fully irrigated. It appears that the production of PR proteins that cause protein instability in wines by grapes may be little influenced by environmental conditions.

  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. Phosphorylation and activation of protamine kinase by two forms of a myelin basic protein kinase from extracts of bovine kidney cortex.

    Science.gov (United States)

    Reddy, S A; Guo, H; Tarun, S Z; Damuni, Z

    1993-07-15

    Two myelin basic protein kinases designated MBPK-1 and MBPK-2 were purified to apparent homogeneity from extracts of bovine kidney cortex. The purified preparations exhibited an apparent M(r) approximately 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 42,000 (MBPK-1) and 45,000 (MBPK-2) by gel permeation chromatography. Up to 0.4 and 1.8 mol of phosphoryl groups were incorporated per mol of MBPK-1 and MBPK-2, respectively, on threonines following incubation with ATP. Autophosphorylation, incubation with protein phosphatase 2A2 (PP2A2), CD45, or T-cell protein tyrosine phosphatase did not affect MBPK-1 activity. Autophosphorylation increased by about 3-fold MBPK-2 activity. This autophosphorylation and activation was reversed by PP2A2 but not by CD45 or T-cell protein tyrosine phosphatase. MBPK-1 and MBPK-2 displayed a positive reaction with an antibody to mitogen-activated protein kinase. Purified preparations of protamine kinase were activated by about 1.5-6-fold and, after inactivation with PP2A2, were reactivated by about 30% by MBPK-1 and MBPK-2. Activation and reactivation correlated with the incorporation, respectively, of 0.1-0.5 and 0.5 mol of phosphoryl groups/mol of the protamine kinase on serines. The results show that MBPK-1 and MBPK-2 are protamine kinase-activating kinases and suggest that MBPK-1 and MBPK-2 may be related to mitogen-activated protein kinase.

  12. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Science.gov (United States)

    Mani, Shailendra; Tripathi, Lav; Raut, Rajendra; Tyagi, Poornima; Arora, Upasana; Barman, Tarani; Sood, Ruchi; Galav, Alka; Wahala, Wahala; de Silva, Aravinda; Swaminathan, Sathyamangalam; Khanna, Navin

    2013-01-01

    Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+)-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (pdengue vaccine.

  13. Modular forms

    NARCIS (Netherlands)

    Edixhoven, B.; van der Geer, G.; Moonen, B.; Edixhoven, B.; van der Geer, G.; Moonen, B.

    2008-01-01

    Modular forms are functions with an enormous amount of symmetry that play a central role in number theory, connecting it with analysis and geometry. They have played a prominent role in mathematics since the 19th century and their study continues to flourish today. Modular forms formed the

  14. Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM

    DEFF Research Database (Denmark)

    Briegel, Ariane; Ortega, Davi R; Mann, Petra

    2016-01-01

    motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed...

  15. Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-28

    The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.

  16. Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

    Science.gov (United States)

    Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

    2005-01-01

    Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

  17. Changes in physical, chemical and functional properties of whey protein isolate (WPI) and sugar beet pectin (SBP) conjugates formed by controlled dry-heating

    Science.gov (United States)

    A Maillard type reaction in the dry state was utilized to create conjugates between whey protein isolate (WPI) and sugar beet pectin (SBP) to achieve improved functional properties including solubility, colloidal stability and oil-in-water emulsion stability. To optimize the reaction conditions, mi...

  18. Membrane-bound and cytosolic forms of heterotrimeric G proteins in young and adult rat myocardium: influence of neonatal hypo- and hyperthyroidism

    Czech Academy of Sciences Publication Activity Database

    Novotný, Jiří; Bouřová, Lenka; Kolář, František; Svoboda, Petr

    2001-01-01

    Roč. 82, č. 2 (2001), s. 215-224 ISSN 0730-2312 R&D Projects: GA ČR GA305/00/1660; GA MŠk VS97099 Institutional research plan: CEZ:AV0Z5011922 Keywords : development * G proteins * young and adult rat myocardium Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.536, year: 2001

  19. Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

    Czech Academy of Sciences Publication Activity Database

    Wald, Tomáš; Osičková, Adriana; Šulc, Miroslav; Benada, Oldřich; Semerádtová, A.; Řežábková, Lenka; Veverka, Václav; Bednárová, Lucie; Malý, J.; Macek, Pavel; Šebo, Peter; Slabý, Ivan; Vondrášek, Jiří; Osička, Radim

    2013-01-01

    Roč. 288, č. 31 (2013), s. 22333-22345 ISSN 0021-9258 R&D Projects: GA ČR GAP302/10/0427 Institutional support: RVO:61388971 ; RVO:61388963 ; RVO:67985823 ; RVO:86652036 Keywords : Ameloblastin * Extracellular Matrix Proteins * Amelogenin Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 4.600, year: 2013

  20. Accumulation of radium in ferruginous protein bodies formed in lung tissue. Association of resulting radiation hotspots with malignant mesothelioma and other malignancies

    International Nuclear Information System (INIS)

    Nakamura, Eizo; Makishima, Akio; Hagino, Kyoko; Okabe, Kazunori

    2009-01-01

    While exposure to fibers and particles has been proposed to be associated with several different lung malignancies including mesothelioma, the mechanism for the carcinogenesis is not fully understood. Along with mineralogical observation, we have analyzed forty-four major and trace elements in extracted asbestos bodies (fibers and proteins attached to them) with coexisting fiber-free ferruginous protein bodies from extirpative lungs of individuals with malignant mesothelioma. These observations together with patients' characteristics suggest that inhaled iron-rich asbestos fibers and dust particles, and excess iron deposited by continuous cigarette smoking would induce ferruginous protein body formation resulting in ferritin aggregates in lung tissue. Chemical analysis of ferruginous protein bodies extracted from lung tissues reveals anomalously high concentrations of radioactive radium, reaching millions of times higher concentration than that of seawater. Continuous and prolonged internal exposure to hotspot ionizing radiation from radium and its daughter nuclides could cause strong and frequent DNA damage in lung tissue, initiate different types of tumour cells, including malignant mesothelioma cells, and may cause cancers. (author)

  1. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Shailendra Mani

    Full Text Available Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4. A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p<0.05. The formation of immunogenic DENV-2 E

  2. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Simon Imhof

    2016-04-01

    Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  3. A chemical approach for site-specific identification of NMR signals from protein side-chain NH3+ groups forming intermolecular ion pairs in protein–nucleic acid complexes

    International Nuclear Information System (INIS)

    Anderson, Kurtis M.; Nguyen, Dan; Esadze, Alexandre; Zandrashvili, Levani; Gorenstein, David G.; Iwahara, Junji

    2015-01-01

    Protein–nucleic acid interactions involve intermolecular ion pairs of protein side-chain and DNA or RNA phosphate groups. Using three protein–DNA complexes, we demonstrate that site-specific oxygen-to-sulfur substitution in phosphate groups allows for identification of NMR signals from the protein side-chain NH 3 + groups forming the intermolecular ion pairs. A characteristic change in their 1 H and 15 N resonances upon this modification (i.e., substitution of phosphate to phosphorodithioate) can represent a signature of an intermolecular ion pair. Hydrogen-bond scalar coupling between protein side-chain 15 N and DNA phosphorodithiaote 31 P nuclei provides direct confirmation of the intermolecular ion pair. The same approach is likely applicable to protein–RNA complexes as well

  4. A chemical approach for site-specific identification of NMR signals from protein side-chain NH{sub 3}{sup +} groups forming intermolecular ion pairs in protein–nucleic acid complexes

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Kurtis M. [University of Texas Health Science Center at Houston, Department of NanoMedicine and Biomedical Engineering and Institute of Molecular Medicine (United States); Nguyen, Dan; Esadze, Alexandre; Zandrashvili, Levani [University of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics (United States); Gorenstein, David G. [University of Texas Health Science Center at Houston, Department of NanoMedicine and Biomedical Engineering and Institute of Molecular Medicine (United States); Iwahara, Junji, E-mail: juiwahar@utmb.edu, E-mail: j.iwahara@utmb.edu [University of Texas Medical Branch, Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics (United States)

    2015-05-15

    Protein–nucleic acid interactions involve intermolecular ion pairs of protein side-chain and DNA or RNA phosphate groups. Using three protein–DNA complexes, we demonstrate that site-specific oxygen-to-sulfur substitution in phosphate groups allows for identification of NMR signals from the protein side-chain NH{sub 3}{sup +} groups forming the intermolecular ion pairs. A characteristic change in their {sup 1}H and {sup 15}N resonances upon this modification (i.e., substitution of phosphate to phosphorodithioate) can represent a signature of an intermolecular ion pair. Hydrogen-bond scalar coupling between protein side-chain {sup 15}N and DNA phosphorodithiaote {sup 31}P nuclei provides direct confirmation of the intermolecular ion pair. The same approach is likely applicable to protein–RNA complexes as well.

  5. Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

    Science.gov (United States)

    Kian, Danielle; Lancheros, César Armando Contreras; Damiani, Igor Alexandre Campos; Fernandes, Tamiris Zanforlin Olmos; Pinge-Filho, Phileno; Santos, Márcia Regina Machado dos; Silveira, José Franco da; Nakamura, Celso Vataru; Silva, João Santana da; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2015-08-01

    This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

  6. A MVA construct expressing a secretable form of the Dengue virus 3 envelope protein protects immunized mice from dengue-induced encephalitis.

    Science.gov (United States)

    Quinan, Bárbara R; Versiani, Alice Freitas; da Fonseca, Flávio G

    2016-12-07

    Dengue is no longer restricted to tropical developing countries, but is now a major global public health problem. Despite the recent license approval of the CYD-TDV vaccine in some countries, efforts to develop a more efficient vaccine against Dengue virus (DENV) continue. Herein, we evaluate the immunogenicity and level of protection of two potential vaccines against DENV based on recombinant modified vaccinia virus Ankara (rMVA). The vaccine addressing the Envelope protein from DENV serotype 3 to the endoplasmic reticulum elicited neutralizing antibodies titers which correlate with protection, and also confers protection upon challenge in a mouse model. Our results support the development of a tetravalent dengue vaccine with the further construction of rMVAs expressing proteins from the other DENV serotypes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbReceptor Family of Proteins

    Science.gov (United States)

    2006-06-01

    higher-order oligomer) can bind to the cholera toxin promoter and activate transcription of a reporter gene. Using this assay, Lemmon and colleagues...identical to the transmembrane sequences of the chicken Klg and Hydra Lemon orthologues (9), and a pattern of residues with helical periodicity is...Steele, R. E. (2000) Lemon encodes an unusual receptor protein-tyrosine kinase expressed during gametogenesis in Hydra , DeV. Biol. 224, 286-298. 10

  8. Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

    OpenAIRE

    Batista, Milene Tavares; Souza, Renata D.; Ferreira, Ewerton L.; Robinette, Rebekah; Crowley, Paula J.; Rodrigues, Juliana F.; Brady, L. Jeannine; Ferreira, Luís C. S.; Ferreira, Rita C. C.

    2014-01-01

    Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P139–512), produced in Bacillus subtilis and encompassing a functional dom...

  9. QGQS Granule in SHR Serum Metabonomics Study Based on Tools of UPLC-Q-TOF and Renin-Angiotensin-Aldosterone System Form Protein Profilin-1

    Directory of Open Access Journals (Sweden)

    Ke Li

    2017-01-01

    Full Text Available QGQS granule is effective for the therapeutic of hypertension in clinic. The aim of this research is to observe the antihypertension effect of QGQS granule on SHR and explain the mechanism of its lowering blood pressure. 30 SHR were selected as model group, captopril group, and QGQS group, 10 WKYr were used as control group, and RBP were measured on tail artery consciously. And all the serum sample analysis was carried out on UPLC-TOF-MS system to determine endogenous metabolites and to find the metabonomics pathways. Meanwhile, ELISA kits for the determination pharmacological indexes of PRA, AngI, AngII, and ALD were used for pathway confirmatory; WB for determination of profilin-1 protein expression was conducted for Ang II pathway analysis as well. It is demonstrated that QGQS granule has an excellent therapeutic effect on antihypertension, which exerts effect mainly on metabonomics pathway by regulating glycerophospholipid, sphingolipid, and arachidonic acid metabolism, and it could inhibit the overexpression of the profilin-1 protein. We can come to a conclusion that RAAS should be responsible mainly for the metabonomics pathway of QGQS granule on antihypertension, and it plays a very important role in protein of profilin-1 inhibition.

  10. The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

    Science.gov (United States)

    Brzovic, Peter S; Heikaus, Clemens C; Kisselev, Leonid; Vernon, Robert; Herbig, Eric; Pacheco, Derek; Warfield, Linda; Littlefield, Peter; Baker, David; Klevit, Rachel E; Hahn, Steven

    2011-12-23

    The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Peptides are building blocks of heat-induced fibrillar protein aggregates of beta-lactoglobulin formed at pH2

    NARCIS (Netherlands)

    Akkermans, C.; Venema, P.; Goot, van der A.J.; Gruppen, H.; Bakx, E.J.; Boom, R.M.; Linden, van der E.

    2008-01-01

    The proteinaceous material present in beta-lactoglobulin fibrils formed after heating (20 h at 85 degrees C) at pH 2 was identified during this study. Fibrils were separated from the nonaggregated material, and the fibrils were dissociated using 8 M guanidine chloride and 0.1 M 1,4-dithiothreitol

  12. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

  13. Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor.

    OpenAIRE

    Lantz, M; Gullberg, U; Nilsson, E; Olsson, I

    1990-01-01

    Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammatory responses. A cysteine-rich, highly glycosylated 30-kD TNF-binding protein (TNF-BP) purified from urine may have a role in regulation because it protects in vitro against the biological effects of TNF. The cytotoxic effect of TNF on the fibrosarcoma cell line WEHI 164 was inhibited by 50% at a 10-fold excess of TNF-BP. The binding of TNF to the receptor was partially reversed after the addition of TNF-BP. Results from biosyn...

  14. Analysis of Imatinib and Sorafenib Binding to p38 Compared with c-Abl and b-Raf Provides Structural Insights for Understanding the Selectivity of Inhibitors Targeting the DFG-Out Form of Protein Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Namboodiri, H.; Bukhtiyarova, M; Ramcharan, J; Karpusas, M; Lee, Y; Springman, E

    2010-01-01

    Protein kinases c-Abl, b-Raf, and p38{alpha} are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38{alpha} inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38{alpha}. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38{alpha} in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

  15. Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease

    Science.gov (United States)

    Berrizbeitia, Mariolga; Figueroa, Milagros; Ward, Brian J.; Rodríguez, Jessicca; Jorquera, Alicia; Figuera, Maria A.; Romero, Leomerys; Ndao, Momar

    2012-01-01

    An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease. PMID:23049572

  16. Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptococcus mutans P1 surface protein.

    Science.gov (United States)

    Batista, Milene Tavares; Souza, Renata D; Ferreira, Ewerton L; Robinette, Rebekah; Crowley, Paula J; Rodrigues, Juliana F; Brady, L Jeannine; Ferreira, Luís C S; Ferreira, Rita C C

    2014-12-01

    Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Prognostic value of inhibitors of apoptosis proteins (IAPs) and caspases in prostate cancer: caspase-3 forms and XIAP predict biochemical progression after radical prostatectomy

    International Nuclear Information System (INIS)

    Rodríguez-Berriguete, Gonzalo; Torrealba, Norelia; Ortega, Miguel Angel; Martínez-Onsurbe, Pilar; Olmedilla, Gabriel; Paniagua, Ricardo; Guil-Cid, Manuel; Fraile, Benito; Royuela, Mar

    2015-01-01

    The expression status of apoptotic regulators, such as caspases and inhibitors of apoptosis proteins (IAPs), could reflect the aggressiveness of tumors and, therefore, could be useful as prognostic markers. We explored the associations between tumor expression of caspases and IAPs and clinicopathological features of prostate cancer – clinical and pathological T stage, Gleason score, preoperative serum PSA levels, perineural invasion, lymph node involvement, surgical margin status and overall survival – and evaluated its capability to predict biochemical progression after radical prostatectomy. Protein expression of caspases (procaspase-8, cleaved caspase-8, procaspase-3, cleaved caspase-3, caspase-7 and procaspase-9) and IAPs (cIAP1/2, cIAP2, NAIP, Survivin and XIAP) was analyzed by immunohistochemistry in radical prostatectomy samples from 84 prostate cancer patients. Spearman’s test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analysis were performed. cIAP1/2, cIAP2, Survivin, procaspase-8, cleaved caspase-8, procaspase-3 and caspase-7 expression correlated with at least one clinicopathological feature of the disease. Patients negative for XIAP, procaspase-3 or cleaved caspase-3 had a significantly worse prognosis. Of note, XIAP, procaspase-3 and cleaved caspase-3 were predictors of biochemical progression independent of Gleason score and pathological T stage. Our results indicate that alterations in the expression of IAPs and caspases contribute to the malignant behavior of prostate tumors and suggest that tumor expression of XIAP, procaspase-3 and cleaved caspase-3 may help to identify prostate cancer patients at risk of progression

  18. Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.

    Science.gov (United States)

    Lopez, Christopher R; Singh, Shivani; Hambarde, Shashank; Griffin, Wezley C; Gao, Jun; Chib, Shubeena; Yu, Yang; Ira, Grzegorz; Raney, Kevin D; Kim, Nayun

    2017-06-02

    G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

    Directory of Open Access Journals (Sweden)

    Joensuu Jussi J

    2009-08-01

    provides an effective strategy for depositing large amounts of concentrated heterologous protein within the limited space of the cell via storage in stable protein bodies. Furthermore, encapsulation of recombinant proteins into physiologically inert organelles can function to insulate the protein from normal cellular mechanisms, thus limiting unnecessary stress to the host cell. Since elastin-like polypeptide is a mammalian-derived protein, this study demonstrates that plant seed-specific factors are not required for the formation of protein bodies in vegetative plant tissues, suggesting that the endoplasmic reticulum possesses an intrinsic ability to form protein body-like accretions in eukaryotic cells when overexpressing particular proteins.

  20. Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

    Science.gov (United States)

    Tao, Tao; Zhou, Cui-Ji; Wang, Qian; Chen, Xiang-Ru; Sun, Qian; Zhao, Tian-Yu; Ye, Jian-Chun; Wang, Ying; Zhang, Zong-Ying; Zhang, Yong-Liang; Guo, Ze-Jian; Wang, Xian-Bing; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2017-01-01

    As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

  1. Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

    Directory of Open Access Journals (Sweden)

    Tao Tao

    Full Text Available As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1 plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA, jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV, a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2 from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

  2. Characterization of the unfolding process of the tetrameric and dimeric forms of Cratylia mollis seed lectin (CRAMOLL 1): effects of natural fragmentation on protein stability.

    Science.gov (United States)

    Varejão, Nathalia; Correia, Maria Tereza S; Foguel, Debora

    2011-08-30

    pCRAMOLL 1 is a major, non-glycosylated isolectin found in seeds of Cratylia mollis, which belongs to the Leguminosae family and the Diocleinae subtribe. The lectin (~25 kDa) consists of 236 amino acids, sharing 82% identity and virtually identical topological architecture with concanavalin A. Both lectins also share the same pH-dependent dimer-tetramer equilibrium and the ability to recognize Glc/Man moieties. Intricate post-translational events occurring in Diocleinae seed cotyledons result in a mixture of intact and fragmented monomers within the oligomeric assemblies of pCRAMOLL 1. In an earlier report, we demonstrated the production, purification, and characterization of the bacterially expressed form of CRAMOLL 1 (rCRAMOLL 1). The recombinant lectin retained sugar-binding activity and several other biophysical properties of pCRAMOLL 1, but its tetramers, which are composed of intact monomers only, show little enhancement in stability when probed with acidification, high temperatures, or hydrostatic pressure. Here we examined the urea-induced unfolding of the nonfragmented tetramers and dimers of rCRAMOLL 1 and compared this behavior with that of the mixed plant lectin counterparts. Using fluorescence, circular dichroism, size-exclusion chromatography, and chemical cross-linking experiments, we posited that the absence of fragmentation lent greater firmness to tetramers, but not to dimers. Dimeric and tetrameric pCRAMOLL 1 unfolded via a compact monomeric intermediate. In contrast, dimers of rCRAMOLL 1 behaved similarly to the plant dimer counterpart, but its tetrameric form remarkably showed no evidence of such partially unfolded monomers. By analyzing the crystal structure of pCRAMOLL 1, we were able to dissect the importance of the fragmentation to lectin stability.

  3. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

    Science.gov (United States)

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

    2016-05-06

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN*

    OpenAIRE

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K.; Smith, Douglas Y.; Söderberg, Christopher A. G.; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

    2016-01-01

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of th...

  5. The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

    Science.gov (United States)

    Ranaghan, Matthew J; Durney, Michael A; Mesleh, Michael F; McCarren, Patrick R; Garvie, Colin W; Daniels, Douglas S; Carey, Kimberly L; Skepner, Adam P; Levine, Beth; Perez, Jose R

    2017-12-26

    Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (K D ∼ 0.45 μM) that interacts with Bcl-2 (K D = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer K D 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

  6. The CCAAT/enhancer binding protein (C/EBP δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-β peptide

    Directory of Open Access Journals (Sweden)

    Nilsson Lars NG

    2011-04-01

    Full Text Available Abstract Background The transcription factors CCAAT/enhancer binding proteins (C/EBP α, β and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB. In general, C/EBPα is down-regulated, whereas both C/EBPβ and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-β (Aβ deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aβ deposits with the aim of defining new therapeutic targets. Methods Here we have investigated the effects of Aβ on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aβ deposits (tg-ArcSwe by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay. Results We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aβ inhibit up-regulation of C/EBPδ expression in interleukin-1β-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPβ was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aβ deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1β-induced C/EBPδ DNA

  7. The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.

    Science.gov (United States)

    Fujikawa, K; Kamiya, H; Yakushiji, H; Fujii, Y; Nakabeppu, Y; Kasai, H

    1999-06-25

    The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.

  8. Non-essential genes form the hubs of genome scale protein function and environmental gene expression networks in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Rosenkrantz, Jesper T; Aarts, Henk; Abee, Tjakko; Rolfe, Matthew D; Knudsen, Gitte M; Nielsen, Maj-Britt; Thomsen, Line E; Zwietering, Marcel H; Olsen, John E; Pin, Carmen

    2013-12-17

    networks of S. Typhimurium. Hubs theoretically confer higher resistance to random mutation but a greater susceptibility to directed attacks, however, we found that genes that formed hubs were dispensable for growth, stress adaptation and virulence, suggesting that evolution favors non-essential genes as main connectors in cellular networks.

  9. Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

    Directory of Open Access Journals (Sweden)

    HORACIO MALDONADO

    2008-01-01

    Full Text Available HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levéis were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

  10. Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

    Directory of Open Access Journals (Sweden)

    Sukhdeep Kumar

    Full Text Available Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD varieties. We establish (a that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT, resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

  11. Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa

    Directory of Open Access Journals (Sweden)

    Wiedmer Stefanie

    2017-01-01

    Full Text Available The genus Eimeria (Apicomplexa, Coccidia provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

  12. Contributors Form

    Directory of Open Access Journals (Sweden)

    Chief Editor

    2016-06-01

    to produce preprints or reprints and translate into languages other than English for sale or free distribution; and 4 the right to republish the work in a collection of articles in any other mechanical or electronic format. We give the rights to the corresponding author to make necessary changes as per the request of the journal, do the rest of the correspondence on our behalf and he/she will act as the guarantor for the manuscript on our behalf. All persons who have made substantial contributions to the work reported in the manuscript, but who are not contributors, are named in the Acknowledgment and have given me/us their written permission to be named. If I/we do not include an Acknowledgment that means I/we have not received substantial contributions from non-contributors and no contributor has been omitted.S NoAuthors' NamesContribution (IJCME Guidelines{1 substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; 2 drafting the article or revising it critically for important intellectual content; and 3 final approval of the version to be published. Authors should meet conditions 1, 2, and 3}.SignatureDate                              Note: All the authors are required to sign independently in this form in the sequence given above. In case an author has left the institution/country and whose whereabouts are not known, the senior author may sign on his/her behalf taking the responsibility.No addition/deletion/ or any change in the sequence of the authorship will be permissible at a later stage, without valid reasons and permission of the Editor.If the authorship is contested at any stage, the article will be either returned or will not be processed for publication till the issue is solved.Maximum up to 4 authors for short communication and up to 6 authors for original article.

  13. Desempenho de bezerros da raça Holandesa alimentados com proteína de soja sólida ou líquida Performance of Holstein calves fed soybean meal protein in solid or liquid form

    Directory of Open Access Journals (Sweden)

    Carlos de Sousa Lucci

    2010-02-01

    Full Text Available Avaliaram-se os efeitos da substituição de metade do total de proteína da dieta na forma sólida (farelo de soja por proteína na forma líquida (leite de soja, com relações sólidos:líquidos de 100,0:0%; 87,5:12,5%; e 75,0:25,0%. Utilizaram-se 24 bezerros machos da raça Holandesa com 60 dias de idade, distribuídos em delineamento em blocos ao acaso, com três dietas à base de concentrado (80% e feno de capim coast-cross (Cynodon dactylon (20%, balanceadas para nitrogênio e energia. Nas dietas com partes líquidas, o intuito foi manter o reflexo de formação da goteira esofagiana no período experimental de 10 meses, no qual os animais foram mantidos em confinamento, sendo abatidos ao atingirem pesos corporais superiores a 400 kg. Em alguns dos animais, dotados de cânulas de rúmen, foi possível detectar neste órgão apenas pequenas quantidades do leite de soja ingerido, confirmando sucesso na formação da goteira esofagiana. Os ganhos de peso diários apresentaram redução linear (1,399; 1,341; e 1,191 kg à medida que foram fornecidas maiores quantidades de leite de soja. As conversões de matéria seca e proteína bruta em ganhos de peso, durante os últimos 60 dias experimentais, pioraram conforme aumentaram as quantidades de proteína na forma líquida. O fornecimento de proteína na forma líquida na dieta não altera os rendimentos de carcaça em bezerros holandeses.The effects were assessed of substituting half the total diet protein in solid form (soybean meal with liquid form (soybean milk at solid:liquid ratios of: 100.0:0% L; 87.5:12.5% L; 75.0:25.0% L. Twenty-four Holstein breed 60-d male calf steers were assigned to a randomized block design, with three concentrate (80% diets and 20% coast-cross (Cynodon dactylon hay, balanced for nitrogen and energy. In the diets with liquid parts, the intention was to keep the functional reflex of the esophageal groove throughout the 10 months of the experimental period, in which

  14. Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex*

    Science.gov (United States)

    Chen, Cuicui; Shi, Zhubing; Zhang, Wenqing; Chen, Min; He, Feng; Zhang, Zhenzhen; Wang, Yicui; Feng, Miao; Wang, Wenjia; Zhao, Yun; Brown, Jerry H.; Jiao, Shi; Zhou, Zhaocai

    2014-01-01

    The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies. PMID:24550388

  15. Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: a form of communication during injury, infection, and cell damage. It is never known how far a controversial finding will go! Dedicated to Ferruccio Ritossa.

    Science.gov (United States)

    De Maio, Antonio

    2011-05-01

    Heat shock proteins (hsp) have been found to play a fundamental role in the recovery from multiple stress conditions and to offer protection from subsequent insults. The function of hsp during stress goes beyond their intracellular localization and chaperone role as they have been detected outside cells activating signaling pathways. Extracellular hsp are likely to act as indicators of the stress conditions, priming other cells, particularly of the immune system, to avoid the propagation of the insult. Some extracellular hsp, for instance Hsp70, are associated with export vesicles, displaying a robust activation of macrophages. We have coined the term Stress Observation System (SOS) for the mechanism for sensing extracellular hsp, which we propose is a form of cellular communication during stress conditions. An enigmatic and still poorly understood process is the mechanism for the release of hsp, which do not contain any consensus secretory signal. The export of hsp appears to be a very complex phenomenon encompassing different alternative pathways. Moreover, extracellular hsp may not come in a single flavor, but rather in a variety of physical conditions. This review addresses some of our current knowledge about the release and function of extracellular hsp, in particular those associated with vesicles.

  16. The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2

    DEFF Research Database (Denmark)

    Bozzetti, M P; Massari, S; Finelli, P

    1995-01-01

    . To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major...

  17. Intracellular localization and interaction of mRNA binding proteins as detected by FRET.

    Science.gov (United States)

    David Gerecht, Pamela S; Taylor, Molly A; Port, J David

    2010-09-15

    A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and

  18. Emery-Dreifuss Muscular Dystrophy-Associated Mutant Forms of Lamin A Recruit the Stress Responsive Protein Ankrd2 into the Nucleus, Affecting the Cellular Response to Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Silvia Angori

    2017-05-01

    Full Text Available Background: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2 is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. Methods: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS release and cell viability. Results: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. Conclusions: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.

  19. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins...

  20. LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

    Science.gov (United States)

    Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

    2009-03-01

    Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

  1. Harmonic Maass forms and mock modular forms

    CERN Document Server

    Bringmann, Kathrin; Ono, Ken

    2017-01-01

    Modular forms and Jacobi forms play a central role in many areas of mathematics. Over the last 10-15 years, this theory has been extended to certain non-holomorphic functions, the so-called "harmonic Maass forms". The first glimpses of this theory appeared in Ramanujan's enigmatic last letter to G. H. Hardy written from his deathbed. Ramanujan discovered functions he called "mock theta functions" which over eighty years later were recognized as pieces of harmonic Maass forms. This book contains the essential features of the theory of harmonic Maass forms and mock modular forms, together with a wide variety of applications to algebraic number theory, combinatorics, elliptic curves, mathematical physics, quantum modular forms, and representation theory.

  2. Harmonic maass forms and mock modular forms

    CERN Document Server

    Bringmann, Kathrin; Ono, Ken

    2017-01-01

    Modular forms and Jacobi forms play a central role in many areas of mathematics. Over the last 10-15 years, this theory has been extended to certain non-holomorphic functions, the so-called "harmonic Maass forms". The first glimpses of this theory appeared in Ramanujan's enigmatic last letter to G. H. Hardy written from his deathbed. Ramanujan discovered functions he called "mock theta functions" which over eighty years later were recognized as pieces of harmonic Maass forms. This book contains the essential features of the theory of harmonic Maass forms and mock modular forms, together with a wide variety of applications to algebraic number theory, combinatorics, elliptic curves, mathematical physics, quantum modular forms, and representation theory.

  3. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  4. On good ETOL forms

    DEFF Research Database (Denmark)

    Skyum, Sven

    1978-01-01

    This paper continues the study of ETOL forms and good EOL forms done by Maurer, Salomaa and Wood. It is proven that binary very complete ETOL forms exist, good synchronized ETOL forms exist and that no propagating or synchronized ETOL form can be very complete.......This paper continues the study of ETOL forms and good EOL forms done by Maurer, Salomaa and Wood. It is proven that binary very complete ETOL forms exist, good synchronized ETOL forms exist and that no propagating or synchronized ETOL form can be very complete....

  5. Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

    OpenAIRE

    Zhu, Z; DeLuca, N A; Schaffer, P A

    1996-01-01

    ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it h...

  6. Modular Forms and Weierstrass Mock Modular Forms

    Directory of Open Access Journals (Sweden)

    Amanda Clemm

    2016-02-01

    Full Text Available Alfes, Griffin, Ono, and Rolen have shown that the harmonic Maass forms arising from Weierstrass ζ-functions associated to modular elliptic curves “encode” the vanishing and nonvanishing for central values and derivatives of twisted Hasse-Weil L-functions for elliptic curves. Previously, Martin and Ono proved that there are exactly five weight 2 newforms with complex multiplication that are eta-quotients. In this paper, we construct a canonical harmonic Maass form for these five curves with complex multiplication. The holomorphic part of this harmonic Maass form arises from the Weierstrass ζ-function and is referred to as the Weierstrass mock modular form. We prove that the Weierstrass mock modular form for these five curves is itself an eta-quotient or a twist of one. Using this construction, we also obtain p-adic formulas for the corresponding weight 2 newform using Atkin’s U-operator.

  7. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs...

  8. Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation

    Czech Academy of Sciences Publication Activity Database

    Eisenreichová, Andrea; Klíma, Martin; Bouřa, Evžen

    2016-01-01

    Roč. 72, č. 11 (2016), s. 799-803 ISSN 2053-230X R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : 14-3-3 proteins * Bmh1 * Bmh2 * crystal structure * phosphopeptide Subject RIV: CE - Biochemistry Impact factor: 0.799, year: 2016

  9. The Myeloid-Related Proteins 8 and 14 Complex, a Novel Ligand of Toll-Like Receptor 4, and Interleukin-1 beta Form a Positive Feedback Mechanism in Systemic-Onset Juvenile Idiopathic Arthritis

    NARCIS (Netherlands)

    Frosch, Michael; Ahlmann, Martina; Vogl, Thomas; Wittkowski, Helmut; Wulffraat, Nico; Foell, Dirk; Roth, Johannes

    Objective. Fever of unknown origin is a diagnostic challenge in children, especially for differentiation of systemic-onset juvenile idiopathic arthritis (systemic-onset JIA) and infectious diseases. We undertook this study to analyze the relevance of myeloid-related proteins (MRPs) 8 and 14,

  10. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  11. A sequence predicted to form a stem–loop is proposed to be required for formation of an RNA–protein complex involving the 3'UTR of beta-subunit F0F1-ATPase mRNA

    Czech Academy of Sciences Publication Activity Database

    Kramarova, T. V.; Antonická, Hana; Houštěk, Josef; Cannon, B.; Nedergaard, J.

    2008-01-01

    Roč. 1777, 7-8 (2008), s. 747-757 ISSN 0005-2728 R&D Projects: GA MZd(CZ) NR7790; GA MŠk(CZ) 1M0520 Grant - others:Univerzita Karlova(CZ) 97807 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATPase * RNA-protein komplex * stem-loop secondary structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.447, year: 2008

  12. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection.

    Science.gov (United States)

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A; Meng, Jihong

    2014-08-30

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11-21 aa contain EV-71-specific antigenic sites, whereas positions 1-5 and 51-100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP₁₆₋₄₃, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP₁₆₋₄₃ is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP₁₆₋₄₃ fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6-43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Manufacturing processes 4 forming

    CERN Document Server

    Klocke, Fritz

    2013-01-01

    This book provides essential information on metal forming, utilizing a practical distinction between bulk and sheet metal forming. In the field of bulk forming, it examines processes of cold, warm and hot bulk forming, as well as rolling and a new addition, the process of thixoforming. As for the field of sheet metal working, on the one hand it deals with sheet metal forming processes (deep drawing, flange forming, stretch drawing, metal spinning and bending). In terms of special processes, the chapters on internal high-pressure forming and high rate forming have been revised and refined. On the other, the book elucidates and presents the state of the art in sheet metal separation processes (shearing and fineblanking). Furthermore, joining by forming has been added to the new edition as a new chapter describing mechanical methods for joining sheet metals. The new chapter “Basic Principles” addresses both sheet metal and bulk forming, in addition to metal physics, plastomechanics and computational basics; ...

  14. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers......, are reported. The aim is to depict how the elucidation of the interplay of structures requires the interplay of methods....

  15. Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+ Plasmid Having Adhesion Strength Comparable to Several Commercial Glues.

    Directory of Open Access Journals (Sweden)

    Chao Liang

    Full Text Available The barnacle is well known for its tenacious and permanent attachment to a wide variety of underwater substrates, which is accomplished by synthesizing, secreting and curing a mixture of adhesive proteins termed "barnacle cement". In order to evaluate interfacial adhesion abilities of barnacle cement proteins, the cp19k homologous gene in Balanus albicostatus (Balcp19k was cloned and expressed in Escherichia coli. Here, we report an intriguing discovery of a gel-like super adhesive aggregation produced by Trx-Balcp19k, a recombinant Balcp19k fusion protein. The Trx-Balcp19k consists of an 18 kDa fragment at the N-terminus, which is encoded by pET-32a(+ plasmid and mainly comprised of a thioredoxin (Trx tag, and Balcp19k at the C-terminus. The sticky aggregation was designated as "Trx-Balcp19k gel", and the bulk adhesion strength, biochemical composition, as well as formation conditions were all carefully investigated. The Trx-Balcp19k gel exhibited strong adhesion strength of 2.10 ± 0.67 MPa, which was approximately fifty folds higher than that of the disaggregated Trx-Balcp19k (40 ± 8 kPa and rivaled those of commercial polyvinyl acetate (PVA craft glue (Mont Marte, Australia and UHU glue (UHU GmbH & Co. KG, Germany. Lipids were absent from the Trx-Balcp19k gel and only a trace amount of carbohydrates was detected. We postulate that the electrostatic interactions play a key role in the formation of Trx-Balcp19k gel, by mediating self-aggregation of Trx-Balcp19k based on its asymmetric distribution pattern of charged amino acids. Taken together, we believe that our discovery not only presents a promising biological adhesive with potential applications in both biomedical and technical fields, but also provides valuable paradigms for molecular design of bio-inspired peptide- or protein-based materials.

  16. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    For example, the structural changes that allowed for allosteric regulation of haemoglobin were re- vealed through structural elucidation of the protein in free and oxygen-bound forms by X-ray crystallography. Following this,. X-ray crystallography has been utilized to study a variety of al- losteric proteins including ATCase. 2.

  17. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  18. Method for forming ammonia

    Science.gov (United States)

    Kong, Peter C.; Pink, Robert J.; Zuck, Larry D.

    2008-08-19

    A method for forming ammonia is disclosed and which includes the steps of forming a plasma; providing a source of metal particles, and supplying the metal particles to the plasma to form metal nitride particles; and providing a substance, and reacting the metal nitride particles with the substance to produce ammonia, and an oxide byproduct.

  19. Mesonic Form Factors

    International Nuclear Information System (INIS)

    Bonnet, Frederic D.R.; Edwards, Robert G.; Felming, George T.; Randal Lewis; David Richards

    2003-01-01

    We have started a program to compute the electromagnetic form factors of mesons. We discuss the techniques used to compute the pion form factor and present preliminary results computed with domain wall valence fermions on MILC asqtad lattices, as well as Wilson fermions on quenched lattices. These methods can easily be extended to rho-to-gamma-pi transition form factors

  20. Isotropy of quadratic forms

    Indian Academy of Sciences (India)

    V. Suresh University Of Hyderabad Hyderabad

    2008-10-31

    Oct 31, 2008 ... Historically, the study of quadratic forms was part of number theory; Minkowski, Siegel, Hasse, Eichler, Kneser and several other mathematicians created a rich arithmetic theory of quadratic forms. V. Suresh University Of Hyderabad Hyderabad. Isotropy of quadratic forms ...

  1. Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

    Science.gov (United States)

    Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

    2017-10-01

    Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

  2. The murine cytomegalovirus immune evasion protein m4/gp34 forms biochemically distinct complexes with class I MHC at the cell surface and in a pre-Golgi compartment.

    Science.gov (United States)

    Kavanagh, D G; Koszinowski, U H; Hill, A B

    2001-10-01

    We have recently demonstrated that the murine CMV (MCMV) gene m4 is an immune evasion gene that protects MCMV-infected targets from some virus-specific CTL clones. m4 encodes m4/gp34, a 34-kDa glycoprotein that binds to major histocompatibility complex class I in the endoplasmic reticulum and forms a detergent-stable complex that is exported to the surface of the cell. To investigate how m4/gp34 promotes CTL evasion, we analyzed the assembly and export of m4/gp34-K(b) complexes. We found that 50-70% of K(b) exported over the course of MCMV infection was m4/gp34 associated. Because these complexes are present at the cell surface, it is possible that m4 mediates CTL evasion by interfering with contact between class I and receptors on the T cell. In addition, we found that K(b) retained by the MCMV immune evasion gene m152 formed a novel type of complex with Endo H-sensitive m4/gp34; these complexes are distinguished from the exported complexes by being stable in 1% digitonin and unstable in 1% Nonidet P-40. Because this association occurs in a pre-Golgi compartment, m4/gp34 might also interfere with Ag presentation by affecting some aspect of class I assembly, such as peptide loading. Although m4/gp34 requires beta(2)-microglobulin to bind class I, there was no significant binding of m4/gp34 to beta(2)-microglobulin in the absence of class I H chain, demonstrating that m4/gp34 forms Nonidet P-40-stable complexes specifically with folded conformations of class I. We conclude that m4/gp34 promotes immune evasion by a novel mechanism involving altered assembly and/or T cell recognition of class I molecules.

  3. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  4. Comparison of the level, distribution and form of disease-associated prion protein in variant and sporadic Creutzfeldt-Jakob diseased brain using conformation-dependent immunoassay and Western blot.

    Science.gov (United States)

    Choi, Young Pyo; Gröner, Albrecht; Ironside, James W; Head, Mark W

    2011-03-01

    Disease-associated prion protein (PrP(Sc)) can be distinguished from the cellular isoform (PrP(C)) by conformation-dependent immunoassay (CDI). This technique exploits the presence of an epitope, accessible in PrP(C), but only unmasked by denaturation in PrP(Sc). In this study, we investigated PrP(Sc) in different brain regions in variant and sporadic Creutzfeldt-Jakob disease (CJD) by using CDI, and directly compared the results with those obtained using the more commonly employed protease digestion and Western blotting. In general, there was good agreement between the results, although there were certain discrepancies in relative abundance when the regional distribution in variant CJD cases was considered. The results largely confirmed the previously described targeting of different brain regions by variant and sporadic CJD. Additionally, the combination of protease digestion and CDI detection demonstrated, for the first time, the presence of PrP(Sc) in variant CJD brains that is susceptible to proteolysis under standard conditions.

  5. Severe forms of food allergy.

    Science.gov (United States)

    Sarinho, Emanuel; Lins, Maria das Graças Moura

    To guide the diagnostic and therapeutic management of severe forms of food allergy. Search in the Medline database using the terms "severe food allergy," "anaphylaxis and food allergy," "generalized urticaria and food allergy," and "food protein-induced enterocolitis syndrome" in the last ten years, searching in the title, abstract, or keyword fields. Food allergy can be serious and life-threatening. Milk, eggs, peanuts, nuts, walnuts, wheat, sesame seeds, shrimp, fish, and fruit can precipitate allergic emergencies. The severity of reactions will depend on associated cofactors such as age, drug use at the onset of the reaction, history and persistence of asthma and/or severe allergic rhinitis, history of previous anaphylaxis, exercise, and associated diseases. For generalized urticaria and anaphylaxis, intramuscular epinephrine is the first and fundamental treatment line. For the treatment in acute phase of food-induced enterocolitis syndrome in the emergency setting, prompt hydroelectrolytic replacement, administration of methylprednisolone and ondansetron IV are necessary. It is important to recommend to the patient with food allergy to maintain the exclusion diet, seek specialized follow-up and, in those who have anaphylaxis, to emphasize the need to carry epinephrine. Severe food allergy may occur in the form of anaphylaxis and food-protein-induced enterocolitis syndrome, which are increasingly observed in the pediatric emergency room; hence, pediatricians must be alert so they can provide the immediate diagnosis and treatment. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  6. Micro metal forming

    CERN Document Server

    2013-01-01

    Micro Metal Forming, i. e. forming of parts and features with dimensions below 1 mm, is a young area of research in the wide field of metal forming technologies, expanding the limits for applying metal forming towards micro technology. The essential challenges arise from the reduced geometrical size and the increased lot size. In order to enable potential users to apply micro metal forming in production, information about the following topics are given: tribological behavior: friction between tool and work piece as well as tool wear mechanical behavior: strength and formability of the work piece material, durability of the work pieces size effects: basic description of effects occurring due to the fact, that the quantitative relation between different features changes with decreasing size process windows and limits for forming processes tool making methods numerical modeling of processes and process chains quality assurance and metrology All topics are discussed with respect to the questions relevant to micro...

  7. Gravitation and quadratic forms

    Science.gov (United States)

    Ananth, Sudarshan; Brink, Lars; Majumdar, Sucheta; Mali, Mahendra; Shah, Nabha

    2017-03-01

    The light-cone Hamiltonians describing both pure ( N = 0) Yang-Mills and N = 4 super Yang-Mills may be expressed as quadratic forms. Here, we show that this feature extends to theories of gravity. We demonstrate how the Hamiltonians of both pure gravity and N = 8 supergravity, in four dimensions, may be written as quadratic forms. We examine the effect of residual reparametrizations on the Hamiltonian and the resulting quadratic form.

  8. Gravitation and quadratic forms

    Energy Technology Data Exchange (ETDEWEB)

    Ananth, Sudarshan [Indian Institute of Science Education and Research,Pune 411008 (India); Brink, Lars [Department of Physics, Chalmers University of Technology,S-41296 Göteborg (Sweden); Institute of Advanced Studies and Department of Physics & Applied Physics,Nanyang Technological University,Singapore 637371 (Singapore); Majumdar, Sucheta [Indian Institute of Science Education and Research,Pune 411008 (India); Mali, Mahendra [School of Physics, Indian Institute of Science Education and Research,Thiruvananthapuram, Trivandrum 695016 (India); Shah, Nabha [Indian Institute of Science Education and Research,Pune 411008 (India)

    2017-03-31

    The light-cone Hamiltonians describing both pure (N=0) Yang-Mills and N=4 super Yang-Mills may be expressed as quadratic forms. Here, we show that this feature extends to theories of gravity. We demonstrate how the Hamiltonians of both pure gravity and N=8 supergravity, in four dimensions, may be written as quadratic forms. We examine the effect of residual reparametrizations on the Hamiltonian and the resulting quadratic form.

  9. PowerForms

    DEFF Research Database (Denmark)

    Brabrand, Claus; Møller, Anders; Ricky, Mikkel

    2000-01-01

    such validation. Today, CGI programmers often use Perl libraries for simple server-side validation or program customized JavaScript solutions for client-side validation. We present PowerForms, which is an add-on to HTML forms that allows a purely declarative specification of input formats and sophisticated...... interdependencies of form fields. While our work may be seen as inspiration for a future extension of HTML, it is also available for CGI programmers today through a preprocessor that translates a PowerForms document into a combination of standard HTML and JavaScript that works on all combinations of platforms...

  10. Cooperative Station History Forms

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Various forms, photographs and correspondence documenting the history of Cooperative station instrumentation, location changes, inspections, and...

  11. Electronic Capitalization Asset Form -

    Data.gov (United States)

    Department of Transportation — National Automated Capitalization Authorization Form used by ATO Engineering Services, Logistics, Accounting for the purpose of identifying and capturing FAA project...

  12. Transient interactions between photosynthetic proteins

    NARCIS (Netherlands)

    Hulsker, Rinske

    2008-01-01

    The biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their affinity, which results in a range from static to transient complexes. Electron transfer complexes,

  13. Mastering HTML5 forms

    CERN Document Server

    Gupta, Gaurav

    2013-01-01

    This tutorial will show you how to create stylish forms, not only visually appealing, but interactive and customized, in order to gather valuable user inputs and information.Enhance your skills in building responsive and dynamic web forms using HTML5, CSS3, and related technologies. All you need is a basic understanding of HTML and PHP.

  14. Hepatitis E: Epidemiological forms

    Indian Academy of Sciences (India)

    In these endemic countries, the disease takes two major epidemiological forms. The form that was recognized first was occurrence of large outbreaks affecting several hundred to several thousand cases of acute hepatitis, usually over a short period of a few weeks. However, it was soon realized that the viral agent was also ...

  15. Yeast Interacting Proteins Database: YNR006W, YHL002W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation; ..., as well as for recycling of Golgi proteins and formation of lumenal membranes Rows with this prey as prey ...1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins dest...degradation, as well as for recycling of Golgi proteins and formation of lumenal membranes

  16. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...... and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function...

  17. Protein Misfolding Diseases.

    Science.gov (United States)

    Hartl, F Ulrich

    2017-06-20

    The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

  18. Protein trapping of nanoparticles

    International Nuclear Information System (INIS)

    Ang, Joo C.; Lin, Jack M.; Yaron, Peter N.; White, John W.

    2009-01-01

    Full text: We have observed the formation of protein-nanoparticle complexes at the air-water interfaces from three different methods of presenting the nanoparticles to proteins. The structures formed resemble the 'protein-nanoparticle corona' proposed by Lynch et al. [1-3) in relation to a possible route for nanoparticle entry into living cells. To do this, the methods of x-ray and neutron reflectivity (with isotopic contrast variation between the protein and nanoparticles) have been used to study the structures formed at the air-water interface of l 3 - casein presented to silica nanoparticle dispersions. Whilst the silica dispersions showed no observable reflectivity, strong signals appear in the reflectivity when protein is present. Drop-wise spreading of a small amount of protein at the air-silica sol interface and presentation of the silica sol to an isolated monomolecular protein film (made by the 'flow-trough' method [4]) gave an immediate signal. Mixing the components in solution only produces a slow response but in all cases a similar structure is formed. The different responses are interpreted in structural and stoichiometric ways.

  19. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested that prot......The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...

  20. Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei.

    Science.gov (United States)

    Dörner, Ursula; Maier, Elke; Benz, Roland

    2004-11-17

    Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.

  1. Forms of Arthritis

    Science.gov (United States)

    ... Today, Linda Saisselin takes joy in holding her new grandson—pain free. Photo courtesy of Linda Saisselin Osteoarthritis (OA) — the form of arthritis typically occurring during middle or old age, this is a joint disease that mostly affects ...

  2. NOAA Form 370 Database

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The data set contains information from submitted NOAA Form 370s, also known as the Fisheries Certificate of Origin, for imported shipments of frozen and/or processed...

  3. MAPS Appraisal Report Form

    CERN Multimedia

    HR Department

    2005-01-01

    As announced in Weekly Bulletin 48/2004, from now onwards, the paper MAPS appraisal report form has been replaced by an electronic form, which is available via EDH (on the EDH desktop under Other Tasks / HR & Training) No changes have been made to the contents of the form. Practical information will be available on the web page http://cern.ch/ais/projs/forms/maps/info.htm, and information meetings will be held on the following dates: 18 January 2005: MAIN AUDITORIUM (500-1-001) from 14:00 to 15:30. 20 January 2005: AB AUDITORIUM II (864-1-D02) from14:00 to 15:30. 24 January 2005: AT AUDITORIUM (30-7-018) from 10:00 to 11:30. Human Resources Department Tel. 73566

  4. Science on form

    International Nuclear Information System (INIS)

    Ishizaka, Shozo; Kato, Yoshihiro; Takaki, Ryuji; Toriwaki, Jun-ichiro

    1987-01-01

    The purpose of the Symposium was to discuss interdisciplinal science aspects of form. 'Form' depends on the material and the changes. But, it is the form that appears evident at once and endures. Form is absorbed from every field as media of information. One part of the work covers the description of non-periodic phenomena, morphogenesis or evolution. Irreducible stubborn facts as diseases or social problems, or whatever else that could not be analyzed are integrally challenged to be systematized by computer simulation. The other part covers the finding of laws for determining how systems behave. Attention should be paid to pattern recognition, image processing and pattern formation. The Symposium proceeded with no parallel sessions, and participants from various fields made exciting discussions in an interdisciplinal atmosphere. (Auth.)

  5. VMS forms Output Tables

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — These output tables contain parsed and format validated data from the various VMS forms that are sent from any given vessel, while at sea, from the VMS devices on...

  6. Forms of Dystonia

    Science.gov (United States)

    ... Special Events Faces of Dystonia Donate Donate Online Membership Find an Event Donor Bill of Rights About Dystonia Symptoms & Diagnosis Forms of Dystonia Genetics Glossary Treatment Find a Doctor Oral Medications Botulinum Neurotoxin Neurosurgery ...

  7. Forms of global governence

    Directory of Open Access Journals (Sweden)

    Maxim V. Kharkevich

    2014-01-01

    Full Text Available Global governance as a concept defines the meaning of contemporary world politics both as a discipline and as reality. Interdependent and globalized world requires governance, and a global government has not been formed yet. The theoretical possibility of global governance without global government is proved and justified. The purpose of this article is to analytically identify possible forms of global governance. Three such forms of global governance are identified: hierarchical, market and network. In a hierarchy the governance is due to the asymmetry of power between the parties. Market control happens via anonymous pricing mechanism. Network, in contrast to the market is characterized by a closer value link between the actors, but unlike the hierarchical relationship actors are free to leave the network. Global governance takes three forms and is being implemented by different actors. To determine the most efficient form of global governance is impossible. Efficiency depends on the match between a form and an object of government. It should be noted that meta governance is likely to remain a monopoly of institutionally strong states in global governance.

  8. Polyphony and verb forms

    Directory of Open Access Journals (Sweden)

    Jelena Rajić

    2012-12-01

    Full Text Available This paper examines some special uses of indicative and subjunctive verb forms in Spanish, which contemporary linguistics explains using the notions of polyphony, evidentials, echoic representation, quotatives, etc. These terms, even though they refer to different characteristics and belong to different theoretical frameworks, share one common feature: they all refer to diverse linguistic forms (discourse markers, linguistic negation, quotatives, echoic utterances, etc. characterized by the presence and interaction of different voices or points of view in one discourse sequence. In this study we are interested in a description of quotative or polyphonic meanings expressed by specific verb forms and tenses, the imperfect and the conditional, and also by indicative forms in subordinate substantive clauses with a negative main verb and by subjunctive forms in subordinate concessive clauses. Our research focuses on the analysis of the linguistic conditions that make possible the evidential use of the conditional, the imperfect and the echoic (metarepresentative interpretation of indicative and subjunctive forms in the above-mentioned contexts. The examples we discuss show that evidential and echoic interpretations are inferential meanings derived from the extralinguistic situation and the knowledge that speakers have of the world.

  9. Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

    Science.gov (United States)

    Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

    2013-01-01

    Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

  10. Comparative waste forms study

    International Nuclear Information System (INIS)

    Wald, J.W.; Lokken, R.O.; Shade, J.W.; Rusin, J.M.

    1980-12-01

    A number of alternative process and waste form options exist for the immobilization of nuclear wastes. Although data exists on the characterization of these alternative waste forms, a straightforward comparison of product properties is difficult, due to the lack of standardized testing procedures. The characterization study described in this report involved the application of the same volatility, mechanical strength and leach tests to ten alternative waste forms, to assess product durability. Bulk property, phase analysis and microstructural examination of the simulated products, whose waste loading varied from 5% to 100% was also conducted. The specific waste forms investigated were as follows: Cold Pressed and Sintered PW-9 Calcine; Hot Pressed PW-9 Calcine; Hot Isostatic Pressed PW-9 Calcine; Cold Pressed and Sintered SPC-5B Supercalcine; Hot Isostatic pressed SPC-5B Supercalcine; Sintered PW-9 and 50% Glass Frit; Glass 76-68; Celsian Glass Ceramic; Type II Portland Cement and 10% PW-9 Calcine; and Type II Portland Cement and 10% SPC-5B Supercalcine. Bulk property data were used to calculate and compare the relative quantities of waste form volume produced at a spent fuel processing rate of 5 metric ton uranium/day. This quantity ranged from 3173 L/day (5280 Kg/day) for 10% SPC-5B supercalcine in cement to 83 L/day (294 Kg/day) for 100% calcine. Mechanical strength, volatility, and leach resistance tests provide data related to waste form durability. Glass, glass-ceramic and supercalcine ranked high in waste form durability where as the 100% PW-9 calcine ranked low. All other materials ranked between these two groupings

  11. Severe forms of food allergy

    Directory of Open Access Journals (Sweden)

    Emanuel Sarinho

    Full Text Available Abstract Objectives: To guide the diagnostic and therapeutic management of severe forms of food allergy. Data sources: Search in the Medline database using the terms “severe food allergy,” “anaphylaxis and food allergy,” “generalized urticaria and food allergy,” and “food protein-induced enterocolitis syndrome” in the last ten years, searching in the title, abstract, or keyword fields. Summary of data: Food allergy can be serious and life-threatening. Milk, eggs, peanuts, nuts, walnuts, wheat, sesame seeds, shrimp, fish, and fruit can precipitate allergic emergencies. The severity of reactions will depend on associated cofactors such as age, drug use at the onset of the reaction, history and persistence of asthma and/or severe allergic rhinitis, history of previous anaphylaxis, exercise, and associated diseases. For generalized urticaria and anaphylaxis, intramuscular epinephrine is the first and fundamental treatment line. For the treatment in acute phase of food-induced enterocolitis syndrome in the emergency setting, prompt hydroelectrolytic replacement, administration of methylprednisolone and ondansetron IV are necessary. It is important to recommend to the patient with food allergy to maintain the exclusion diet, seek specialized follow-up and, in those who have anaphylaxis, to emphasize the need to carry epinephrine. Conclusion: Severe food allergy may occur in the form of anaphylaxis and food-protein-induced enterocolitis syndrome, which are increasingly observed in the pediatric emergency room; hence, pediatricians must be alert so they can provide the immediate diagnosis and treatment.

  12. Interfacial Protein-Protein Associations

    OpenAIRE

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2013-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for ...

  13. Chemical forms of radioiodine

    International Nuclear Information System (INIS)

    Tachikawa, Enzo

    1979-01-01

    Release of radioiodine built-up during reactor operations presents a potential problem from the standpoint of environmental safety. Among the chemical forms of radioiodine, depending upon the circumstances, organic iodides cast a most serious problem because of its difficulties in the trapping and because of its stability compared to other chemical forms. Furthermore, pellet-cladding interaction (PCl) fuel failures in LWR fuel rods are believed to be stress corrosion cracks caused by embrittling fission product species, radioiodine. To deal with these problems, knowledge is required on the chemical behaviors of radioiodine in and out of fuels, as well as the release behaviors from fuels. Here a brief review is given of these respects, in aiming at clearing-up the questions still remaining unknown. The data seem to indicate that radioiodine exists as a combined form in fuels. upon heating slightly irradiated fuels, the iodine atoms are released in a chemical form associated with uranium atoms. Experiments, however, as needed with specimen of higher burnup, where the interactions of radioiodine with metallic fission products could be favored. The dominant release mechanism of radioiodine under normal operating temperatures will be diffusion to grain boundaries leading to open surfaces. Radiation-induced internal traps, however, after the rate of diffusion significantly. The carbon sources of organic iodides formed under various conditions and its formation mechanisms have also been considered. (author)

  14. Pore-forming toxins in Cnidaria.

    Science.gov (United States)

    Podobnik, Marjetka; Anderluh, Gregor

    2017-12-01

    The ancient phylum of Cnidaria contains many aquatic species with peculiar lifestyle. In order to survive, these organisms have evolved attack and defense mechanisms that are enabled by specialized cells and highly developed venoms. Pore-forming toxins are an important part of their venomous arsenal. Along some other types, the most representative are examples of four protein families that are commonly found in other kingdoms of life: actinoporins, Cry-like proteins, aerolysin-like toxins and MACPF/CDC toxins. Some of the homologues of pore-forming toxins may serve other functions, such as in food digestion, development and response against pathogenic organisms. Due to their interesting physico-chemical properties, the cnidarian pore-forming toxins may also serve as tools in medical research and nanobiotechnological applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. On Growth and Form

    Science.gov (United States)

    Maartens, Aidan

    2017-12-01

    D'Arcy Thompson was born in 1860, trained in Edinburgh and Cambridge, and held positions in Dundee and St Andrews, where he worked until his death in 1948. On Growth and Form , his classic work on the mathematical patterns and physical rules underlying biological forms, was first published in 1917. To learn more about the book's context, we met Matthew Jarron, Curator of Museum Services at the University of Dundee, in the University's D'Arcy Thompson Zoology Museum. Surrounded by specimens, many of which were collected by Thompson himself, we discussed the legacy of On Growth and Form and the life of the man behind it. © 2017. Published by The Company of Biologists Ltd.

  16. Waste-form development

    International Nuclear Information System (INIS)

    Neilson, R.M. Jr.; Colombo, P.

    1982-01-01

    Contemporary solidification agents are being investigated relative to their applications to major fuel cycle and non-fuel cycle low-level waste (LLW) streams. Work is being conducted to determine the range of conditions under which these solidification agents can be applied to specific LLW streams. These studies are directed primarily towards defining operating parameters for both improved solidification of problem wastes and solidification of new LLW streams generated from advanced volume reduction technologies. Work is being conducted to measure relevant waste form properties. These data will be compiled and evaluated to demonstrate compliance with waste form performance and shallow land burial acceptance criteria and transportation requirements

  17. Sixth form pure mathematics

    CERN Document Server

    Plumpton, C

    1968-01-01

    Sixth Form Pure Mathematics, Volume 1, Second Edition, is the first of a series of volumes on Pure Mathematics and Theoretical Mechanics for Sixth Form students whose aim is entrance into British and Commonwealth Universities or Technical Colleges. A knowledge of Pure Mathematics up to G.C.E. O-level is assumed and the subject is developed by a concentric treatment in which each new topic is used to illustrate ideas already treated. The major topics of Algebra, Calculus, Coordinate Geometry, and Trigonometry are developed together. This volume covers most of the Pure Mathematics required for t

  18. Prokaryotic Ubiquitin-Like Protein Modification

    OpenAIRE

    Maupin-Furlow, Julie A.

    2014-01-01

    Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier protein...

  19. Acanthamoeba castellanii STAT protein.

    Science.gov (United States)

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  20. Acanthamoeba castellanii STAT protein.

    Directory of Open Access Journals (Sweden)

    Anna Kicinska

    Full Text Available STAT (signal transducers and activators of transcription proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil, a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  1. Many Forms of Culture

    Science.gov (United States)

    Cohen, Adam B.

    2009-01-01

    Psychologists interested in culture have focused primarily on East-West differences in individualism-collectivism, or independent-interdependent self-construal. As important as this dimension is, there are many other forms of culture with many dimensions of cultural variability. Selecting from among the many understudied cultures in psychology,…

  2. Sesotho Address Forms

    Directory of Open Access Journals (Sweden)

    Akindele, Dele Femi

    2008-01-01

    Full Text Available Address forms constitute an integral part of Basotho sociolinguistic etiquette. They are regarded as a kind of emotional capital that may be invested in putting others at ease. They are indicators of deference, politeness and markers of social distance. (Fasold 1990, Akindele 1990, 1991, 1993 This paper examines the address forms used by the Basotho people. It analyzes and discusses the various types and the factors determining their use. The discussion of address forms in Sesotho focuses on First Name, Title plus First Name, Title plus Last Name, Nickname, Multiple Names, and Teknonym. Drawing data from semi-literate and literate urban and rural population of Maseru district of Lesotho, it was found that the commonest form of address used by the Basotho people is title plus first name. e.g. ntate Thabo (father Thabo, 'm'e Puleng (mother Puleng, ausi Maneo (sister Maneo, abuti Mahao (brother Mahao. It is used by close relations, associates, and familiar people in both formal and informal situations.

  3. Documentary form no. 1

    International Nuclear Information System (INIS)

    2002-12-01

    This first documentary form, edited by the national association of local commissions of information about nuclear activities (ANCLI), briefly presents the radioactivity phenomenon, the ionising radiations, the characteristics of radiation sources (activity, half life, energy), and the dosimetry (absorbed, equivalent, efficient doses). (J.S.)

  4. Getting in-formed

    DEFF Research Database (Denmark)

    Hansbøl, Mikala

    Work-in-progress paper præsenteret ved PhD Seminar: Analytical strategies and methodologies for the study of virtual worlds at RUC. September 28th to October 1st 2009. I paperet diskuteres den dobbelte bevægelse at vi som forskere via vores undersøgelser bliver informeret og i samme moment putter...... det vi undersøger på form gennem vores beskrivelser. Paperet tager afsæt i empiriske eksempler fra et postdoc projekt om et såkaldt 'serious game' - Mingoville. Projektet følger circuleringer og etableringer af Mingoville 'på en global markedsplads'. I paperet diskuteres hvordan vi som forskere samler....../performer de fænomener vi forsker i. Aktør-Netværks-Teoretiker Bruno Latour (2005) pointerer at enhver beskrivelse også er en form for forklaring. En form for forklaring, der putter ting ind i et skript og dermed også putter ting på form. Paperet diskuterer to tilgange til at gøre serious games og derved skabe viden om...

  5. Manifolds admitting stable forms

    Czech Academy of Sciences Publication Activity Database

    Le, Hong-Van; Panák, Martin; Vanžura, Jiří

    2008-01-01

    Roč. 49, č. 1 (2008), s. 101-11 ISSN 0010-2628 R&D Projects: GA ČR(CZ) GP201/05/P088 Institutional research plan: CEZ:AV0Z10190503 Keywords : stable forms * automorphism groups Subject RIV: BA - General Mathematics

  6. Inequalities for Differential Forms

    CERN Document Server

    Agarwal, Ravi P

    2009-01-01

    Presents a series of local and global estimates and inequalities for differential forms, in particular the ones that satisfy the A-harmonic equations. This work focuses on the Hardy-Littlewood, Poincare, Cacciooli, imbedded and reverse Holder inequalities. It is for researchers, instructors and graduate students

  7. Metal forming and lubrication

    DEFF Research Database (Denmark)

    Bay, Niels

    2000-01-01

    Lubrication is essential in most metal forming processes. The lubricant film has two basic functions, [1]: i. to separate the work piece and tool surfaces and ii. to cool the workpiece and the tool. Separation of the two surfaces implies lower friction facilitating deformation and lowering the tool...

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

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... epilepsy, ulcers, and eye diseases in China for thousands of years. It has also been used for the prevention of ..... medium of Drosophila wing-disc C1.8+ cells with the ability to stimulate the proliferation, .... family of growth factors produced by the fat body and active on. Drosophila imaginal disc cells.

  9. Protein misfolding disorders: pathogenesis and intervention

    DEFF Research Database (Denmark)

    Gregersen, Niels

    2006-01-01

    protein is eliminated by one of the protein quality control systems. Examples are cystic fibrosis and phenylketonuria. However, not all aberrant proteins can be eliminated and the misfolded protein may accumulate and form toxic oligomeric and/or aggregated inclusions. In this case the loss of function may...

  10. Moonlighting proteins : An intriguing mode of multitasking

    NARCIS (Netherlands)

    Huberts, Daphne; van der Klei, I.J.

    Proteins are macromolecules, which perform a large variety of functions. Most of them have only a single function, but an increasing number of proteins are being identified as multifunctional. Moonlighting proteins form a special class of multifunctional proteins. They perform multiple autonomous

  11. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  12. Modeling Mercury in Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Parks, Jerry M [ORNL

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  13. Yeast Interacting Proteins Database: YHL002W, YNR006W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ycling of Golgi proteins and formation of lumenal membranes Rows with this bait as bait (1) Rows with this b...required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined...on, as well as for recycling of Golgi proteins and formation of lumenal membranes...ith Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated protei

  14. Waste form development

    International Nuclear Information System (INIS)

    Neilson, R.M. Jr.; Colombo, P.

    1982-01-01

    In this program, contemporary solidification agents are being investigated relative to their applications to major fuel cycle and non-fuel cycle low-level waste (LLW) streams. Work is being conducted to determine the range of conditions under which these solidification agents can be applied to specific LLW streams. These studies are directed primarily towards defining operating parameters for both improved solidification of problem wastes and solidification of new LLW streams generated from advanced volume reduction technologies. Work is being conducted to measure relevant waste form properties. These data will be compiled and evaluated to demonstrate compliance with waste form performance and shallow land burial acceptance criteria and transportation requirements (both as they exist and as they are modified with time). 6 tables

  15. Formed HIP Can Processing

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, Kester Diederik [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-07-27

    The intent of this report is to document a procedure used at LANL for HIP bonding aluminum cladding to U-10Mo fuel foils using a formed HIP can for the Domestic Reactor Conversion program in the NNSA Office of Material, Management and Minimization, and provide some details that may not have been published elsewhere. The HIP process is based on the procedures that have been used to develop the formed HIP can process, including the baseline process developed at Idaho National Laboratory (INL). The HIP bonding cladding process development is summarized in the listed references. Further iterations with Babcock & Wilcox (B&W) to refine the process to meet production and facility requirements is expected.

  16. Forms of Inattentiveness

    DEFF Research Database (Denmark)

    Knudsen, Morten

    2011-01-01

    and kept out of sight in the decision processes by looking at a specific case study involving the construction of a model intended to control, and render transparent, the quality of health services in Denmark. This paper outlines the forms of inattentiveness which make communication blind to information...... that could question the quality model. Five forms of inattentiveness are identified that function as answers to the question of how communication avoids actualizing relevant but also potentially destructive information. This study documents a considerable amount of blindness to potentially relevant themes...... effect of the categories applied. This paper puts forward a fourth type of organizational blindness in addition to the already documented ones, namely self-imposed blindness to potentially destructive information. This paper studies how relevant - but problematic - information is actively ignored...

  17. Differential forms of supermanifolds

    International Nuclear Information System (INIS)

    Beresin, P.A.

    1979-01-01

    The theory of differential and pseUdo-differential forms on supermanifolds is constructed. The definition and notations of superanalogy of the Pontryagin and Chern characteristic classes are given. The theory considered is purely local. The scheme suggested here generalizes the so-called Weil homomorphism for superspace which lays on the basis of the Chern and Potryagin characteristic class theory. The theory can be extended to the global supermanifolds

  18. Personal Information Request Form

    International Development Research Centre (IDRC) Digital Library (Canada)

    PC Forms Inc. 834-4048

    Request Form. Head Office / Siège : PO Box / CP 8500, Ottawa, ON, Canada K1G 3H9 www.idrc.ca www.crdi.ca. (150 Kent Street, Constitution Square / 150, rue Kent, Complexe Constitution Square). Phone / Tél. : 613-236-6163, ext./poste 2348 Fax / Téléc. : 613-565-8212 Email / Courriel : ATIP-AIPRP@idrc.ca. Privacy Act.

  19. Forms of Life

    Directory of Open Access Journals (Sweden)

    Peter Hacker

    2015-10-01

    Full Text Available The phrase ‘Lebensform’ (form of life had a long and varied history prior to Wittgenstein’s use of it on a mere three occasions in the Philosophical Investigations. It is not a pivotal concept in Wittgenstein’s philosophy. But it is a minor signpost of a major reorientation of philosophy, philosophy of language and logic, and philosophy of mathematics that Wittgenstein instigated. For Wittgenstein sought to replace the conception of a language as a meaning calculus (Frege, Russell, the Tractatus by an anthropological or ethnological conception. A language is not a class of sentences that can be formed from a set of axioms (definitions, formation and transformation rules and the meanings of which is given by their truth-conditions, but an open-ended series of interlocking language-games constituting a form of life or way of living (a culture. Wittgenstein’s uses of ‘Lebensform’ and its cognates, both in the Investigations and in his Nachlass are severally analysed, and various exegetical misinterpretations are clarified.

  20. Alternative proteins: A New Green Revolution: Dietary Proteins From Leaves

    NARCIS (Netherlands)

    Geerdink, P.; Diaz, J.; Jong, J. de; Bussmann, P.

    2017-01-01

    The fractionation and isolation of leaf proteins, mostly in the form of a photosynthetic enzyme, RuBisCO, contributes to improving sustainability and increasing profitability for the agro-industrial sector.

  1. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  2. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  3. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  4. Electron transfer in proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1991-01-01

    Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable...

  5. Dark proteins disturb multichromophore coupling in tetrameric fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J.; Subramaniam, Vinod

    2011-01-01

    DsRed is representative of the tetrameric reef coral fluorescent proteins that constitute particularly interesting coupled multichromophoric systems. Either a green emitting or a red emitting chromophore can form within each of the monomers of the protein tetramer. Within the tetramers the

  6. Bipolar pulse forming line

    Science.gov (United States)

    Rhodes, Mark A.

    2008-10-21

    A bipolar pulse forming transmission line module for linear induction accelerators having first, second, third, fourth, and fifth planar conductors which form an interleaved stack with dielectric layers between the conductors. Each conductor has a first end, and a second end adjacent an acceleration axis. The first and second planar conductors are connected to each other at the second ends, the fourth and fifth planar conductors are connected to each other at the second ends, and the first and fifth planar conductors are connected to each other at the first ends via a shorting plate adjacent the first ends. The third planar conductor is electrically connectable to a high voltage source, and an internal switch functions to short a high voltage from the first end of the third planar conductor to the first end of the fourth planar conductor to produce a bipolar pulse at the acceleration axis with a zero net time integral. Improved access to the switch is enabled by an aperture through the shorting plate and the proximity of the aperture to the switch.

  7. TRANSIENT AMORPHOUS CALCIUM PHOSPHATE IN FORMING ENAMEL

    OpenAIRE

    Beniash, Elia; Metzler, Rebecca A.; Lam, Raymond S.K.; Gilbert, P.U.P.A.

    2009-01-01

    Enamel, the hardest tissue in the body, begins as a three-dimensional network of nanometer size mineral particles, suspended in a protein gel. This mineral network serves as a template for mature enamel formation. To further understand the mechanisms of enamel formation we characterized the forming enamel mineral at an early secretory stage using x-ray absorption near-edge structure (XANES) spectromicroscopy, transmission electron microscopy (TEM), FTIR microspectroscopy and polarized light m...

  8. Protein-Protein Interaction on Lysozyme Crystallization Revealed by Rotational Diffusion Analysis

    OpenAIRE

    Takahashi, Daisuke; Nishimoto, Etsuko; Murase, Tadashi; Yamashita, Shoji

    2008-01-01

    Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Usi...

  9. Pion form factor

    Energy Technology Data Exchange (ETDEWEB)

    Ryong Ji, C.; Pang, A.; Szczepaniak, A. [North Carolina State Univ., Raleigh, NC (United States)

    1994-04-01

    It is pointed out that the correct criterion to define the legal PQCD contribution to the exclusive processes in the lightcone perturbative expansion should be based on the large off-shellness of the lightcone energy in the intermediate states. In the lightcone perturbative QCD calculation of the pion form factor, the authors find that the legal PQCD contribution defined by the lightcone energy cut saturates in the smaller Q{sup 2} region compared to that defined by the gluon four-momentum square cut. This is due to the contribution by the highly off-energy-shell gluons in the end point regions of the phase space, indicating that the gluon four-momentum-square cut may have cut too much to define the legal PQCD.

  10. Image forming apparatus

    DEFF Research Database (Denmark)

    2005-01-01

    An image H(x, y) for displaying a target image G(x, y) is displayed on a liquid-crystal display panel and illumination light from an illumination light source is made to pass therethrough to form an image on a PALSLM. Read light hv is radiated to the PALSLM and a phase-modulated light image alpha...... (x, y) read out of the PALSLM is subjected to Fourier transform by a lens. A phase contrast filter gives a predetermined phase shift to only the zero-order light component of Fourier light image alpha f(x, y). The phase-shifted light image is subjected to inverse Fourier transform by a lens...... characteristics of the illumination light source, PALSLM, and phase contrast filter, based on the evaluation result....

  11. ELEMENTAL FORMS OF HOSPITALITY

    Directory of Open Access Journals (Sweden)

    Maximiliano Emanuel Korstanje

    2010-11-01

    Full Text Available Modern studies emphasized on the needs of researching the hospitality as relevant aspects of tourism and hospitality fields. Anyway, these approaches are inextricably intertwined to the industry of tourism and do not take seriously the anthropological and sociological roots of hospitality. In fact, the hotel seems to be a partial sphere of hospitality at all. Under this context, the present paper explores the issue of hospitality enrooted in the political and economic indo-European principle of free-transit which is associated to a much broader origin.  Starting from the premise etymologically hostel and hospital share similar origins, we follow the contributions of J Derrida to determine the elements that formed the hospitality up to date.

  12. Le forme del fondo

    Directory of Open Access Journals (Sweden)

    Michel Maffesoli

    2004-12-01

    Full Text Available Non è vero che la natura ha paura del vuoto. Forse addirittura si completa. Il vuoto è anche una modalità dell'essere. È possibile nidificarvisi, avvolgersi pigramente e, così, proteggersi dall'angoscia del tempo che passa. Il vuoto delle apparenze è, in alcuni momenti, una delle forme d'espressione della vita sociale. Oltretutto occorre saperle riconoscere. Certamente, abbiamo tutti un'esistenza personale, ma siamo, ugualmente, i rappresentanti, a volte anche le vittime, di uno "spirito comune", forse anche di un "inconscio collettivo" che si è costituito di secolo in secolo. E, molto spesso, quando crediamo di esprimere le nostre idee, siamo soltanto dei portavoce, comparse di un vasto "theatrum mundi" dalle dimensioni infinite.

  13. Dimeric form of peroxynitrite

    Science.gov (United States)

    Simon, K. V.; Tulub, A. V.

    2009-07-01

    The (ONOO)- anion, known as peroxynitrite, is characterized by a singlet spin state. A new stable dimer form of peroxynitrite [NO-O2]- in the triplet state at distances close to r(O-N) ≈ 2.885 Å between oxygen in the O2 structure and nitrogen has been established within the quantum-chemical CASSCF approximation. The vibrational motion of the dimer is significantly anharmonic; for the 16O and 14N isotopes, the differences in the energies of two neighboring levels correspond to frequencies of about 70-30 cm-1. The triplet dimer structure retains stability in the case of interaction with water molecules. The activation barriers caused by rearrangement of the peroxynitrite structure into the ground state of the NO{3/-} anion with the symmetry D 3 h are found.

  14. Nucleon Electromagnetic Form Factors

    Energy Technology Data Exchange (ETDEWEB)

    Marc Vanderhaeghen; Charles Perdrisat; Vina Punjabi

    2007-10-01

    There has been much activity in the measurement of the elastic electromagnetic proton and neutron form factors in the last decade, and the quality of the data has greatly improved by performing double polarization experiments, in comparison with previous unpolarized data. Here we review the experimental data base in view of the new results for the proton, and neutron, obtained at JLab, MAMI, and MIT-Bates. The rapid evolution of phenomenological models triggered by these high-precision experiments will be discussed, including the recent progress in the determination of the valence quark generalized parton distributions of the nucleon, as well as the steady rate of improvements made in the lattice QCD calculations.

  15. Moonlighting proteins: An intriguing mode of multitasking

    OpenAIRE

    Huberts, Daphne; van der Klei, I.J.

    2010-01-01

    Proteins are macromolecules, which perform a large variety of functions. Most of them have only a single function, but an increasing number of proteins are being identified as multifunctional. Moonlighting proteins form a special class of multifunctional proteins. They perform multiple autonomous and often unrelated functions without partitioning these functions into different domains of the protein. Striking examples are enzymes, which in addition to their catalytic function are involved in ...

  16. Recombinant protein hydrazides: application to site-specific protein PEGylation.

    Science.gov (United States)

    Thom, Jennifer; Anderson, David; McGregor, Joanne; Cotton, Graham

    2011-06-15

    Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.

  17. Moon (Form-Origin)

    Science.gov (United States)

    Tsiapas, Elias; Soumelidou, Despina; Tsiapas, Christos

    2017-04-01

    When the Earth was formed, it was in a state of burning heat. As time went by, temperature on the planet's surface was falling due to radiation and heat transfer, and various components (crusts) began taking solid form at the Earth's poles. The formation of crusts took place at the Earth's poles, because the stirring of burning and fluid masses on the surface of the Earth was significantly slighter there than it was on the equator. Due to centrifugal force and Coriolis Effect, these solid masses headed towards the equator; those originating from the North Pole followed a south-western course, while those originating from the South Pole followed a north-western course and there they rotated from west to east at a lower speed than the underlying burning and liquid earth, because of their lower initial linear velocity, their solid state and inertia. Because inertia is proportional to mass, the initially larger solid body swept all new solid ones, incorporating them to its western side. The density of the new solid masses was higher, because the components on the surface would freeze and solidify first, before the underlying thicker components. As a result, the western side of the initial islet of solid rocks submerged, while the east side elevated. . As a result of the above, this initial islet began to spin in reverse, and after taking on the shape of a sphere, it formed the "heart" of the Moon. The Moon-sphere, rolling on the equator, would sink the solid rocks that continued to descend from the Earth's poles. The sinking rocks partially melted because of higher temperatures in the greater depths that the Moon descended to, while part of the rocks' mass bonded with the Moon and also served as a heat-insulating material, preventing the descended side of the sphere from melting. Combined with the Earth's liquid mass that covered its emerging eastern surface, new sphere-shaped shells were created, with increased density and very powerful structural cohesion. During the

  18. Cavitation During Superplastic Forming

    Directory of Open Access Journals (Sweden)

    John Campbell

    2011-07-01

    Full Text Available Cavitation is the opening of pores during superplastic forming, typically at grain boundary triple points or on second phase grain boundary particles during slip of grain boundaries. Theories for the initiation of cavitation are reviewed. It seems that cavitation is unlikely to occur by processes intrinsic to metals such as dislocation mechanisms or point defect condensation. It is proposed that cavitation can only occur at non-bonded interfaces such as those introduced extrinsically (i.e., from the outside during the original casting of the metal. These defects, known as oxide bifilms, are naturally introduced during pouring of the liquid metal, and are frozen into the solid, often pushed by dendritic growth into grain boundaries where they are difficult to detect because of their extreme thinness, often measured in nanometres. Their unbonded central interface acts as a crack and can initiate cavitation. Second phase precipitates probably do not nucleate and grow on grain boundaries but grow on bifilms in the boundaries, explaining the apparent association between boundaries, second phase particles and failure initiation. Improved melting and casting techniques can provide metal with reduced or zero bifilm population for which cavitation would not be possible, promising significant improvements in superplastic behaviour.

  19. Les formes du fond

    Directory of Open Access Journals (Sweden)

    Michel Maffesoli

    2004-12-01

    Full Text Available Il n'est pas vrai que la nature a horreur du vide. Peut-être même s'y complait-elle. Le creux est aussi une modalité de l'être. Il est possible de s'y nicher, de s'y lover paresseusement et, ainsi, de se protéger contre l'angoisse du temps qui passe. Le creux des apparences est, à certains moments, une des formes d'expression de la vie sociale. Encore faut-il savoir le reconnaître. Certes, nous avons tous une existence personnelle, mais nous sommes, également, les représentants, parfois même les victimes, d'un "esprit commun", peut-être même d'un "inconscient collectif" qui s'est constitué de siècle en siècle. Et, très souvent, là où nous croyons exprimer nos propres idées, nous ne sommes que les porte-voix, les figurants d'un vaste "theatrum mundi" aux dimensions infinies.

  20. Image forming apparatus

    DEFF Research Database (Denmark)

    2005-01-01

    An image H(x, y) for displaying a target image G(x, y) is displayed on a liquid-crystal display panel and illumination light from an illumination light source is made to pass therethrough to form an image on a PALSLM. Read light hv is radiated to the PALSLM and a phase-modulated light image alpha...... (x, y) read out of the PALSLM is subjected to Fourier transform by a lens. A phase contrast filter gives a predetermined phase shift to only the zero-order light component of Fourier light image alpha f(x, y). The phase-shifted light image is subjected to inverse Fourier transform by a lens...... to project an output image O(x, y) to an output plane. A light image O'(x, y) branched by a beam sampler is picked up by a pickup device and an evaluation value calculating unit evaluates conformity between the image O(x, y) and the image G(x, y).; A control unit performs feedback control of optical...

  1. Protein Extractability

    African Journals Online (AJOL)

    Results showed that protein extractability was dependent on pH, type of salt, salt concentrations and extraction time. Salts extracted more proteins from the moringa seed flour than water. Maximum extraction of protein was. 85.06% and 84.72% with 0.5 M CaCl and 0.75 M NaCl respectively. On varying the pH, maximum ...

  2. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...... of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode....

  3. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

    Science.gov (United States)

    Pahrudin Arrozi, Aslina; Shukri, Siti Nur Syazwani; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Ahmad Damanhuri, Mohd Hanafi; Makpol, Suzana

    2017-11-01

    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

  4. Forms of War

    International Nuclear Information System (INIS)

    Vogel, H.; Bartelt, D.

    2007-01-01

    Purpose: Under war conditions, employed weapons can be identified on radiographs obtained in X-ray diagnostic. The analysis of such X-ray films allows concluding that there are additional information about the conditions of transport and treatment; it shall be shown that there are X-ray findings which are typical and characteristic for certain forms of warfare. Material and method: The radiograms have been collected during thirty years; they come from hospitals, where war casualties had been treated, and personal collections. Results: The material is selected, because in war X-ray diagnostic will be limited and the interest of the opposing parties influence the access to the material; furthermore the possibilities to publish or to communicate facts and thoughts are different. Citizens of the USA, GB, France, or Israel will have easier access to journals than those of Vietnam, Chad, and Zimbabwe. Under war conditions, poor countries, like North Vietnam may develop own concepts of medical care. There are X-ray findings which are typical or even characteristic for air warfare, guerrilla warfare, gas war, desert warfare, conventional warfare, and annihilation warfare, and city guerrilla warfare/civil war. The examples demonstrate that weapons and the conditions of transport and treatment can be recognized by X-ray findings. The radiogram can be read like a document. Conclusion: In War, there are differences between a treatment and imaging diagnostic in countries, which control the air space and in those who do not. Medical care of the poor, i.e. in countries (in general those opposing the western nations) will hardly be published, and poverty has no advocate

  5. Protein Supplements: Pros and Cons.

    Science.gov (United States)

    Samal, Jay Rabindra Kumar; Samal, Indira R

    2018-05-04

    To provide a comprehensive analysis of the literature examining the pros and cons of protein supplementation, various articles on protein supplementation were obtained from Google Scholar, PubMed, and National Center for Biotechnology Information. Over the past few years, protein supplementation has become commonplace for gym-goers as well as for the public. A large segment of the general population relies on protein supplementation for meal replacement, weight reduction, and purported health benefits. These protein supplements have varying pros and cons associated with them, which are often overlooked by the public. This review aims to assimilate existing studies and form a consensus regarding the benefits and disadvantages of protein supplementation. The purported health benefits of protein supplementation have led to overuse by both adults and adolescents. Although the pros and cons of protein supplementation is a widely debated topic, not many studies have been conducted regarding the same. The few studies that exist either provide insufficient evidence or have not employed proper conditions for the conduct of the tests. It should be considered that protein supplements are processed materials and often do not contain other essential nutrients required for the sustenance of a healthy lifestyle. It is suggested that the required protein intake should be obtained from natural food sources and protein supplementation should be resorted to only if sufficient protein is not available in the normal diet.

  6. Moonlighting proteins: an intriguing mode of multitasking.

    Science.gov (United States)

    Huberts, Daphne H E W; van der Klei, Ida J

    2010-04-01

    Proteins are macromolecules, which perform a large variety of functions. Most of them have only a single function, but an increasing number of proteins are being identified as multifunctional. Moonlighting proteins form a special class of multifunctional proteins. They perform multiple autonomous and often unrelated functions without partitioning these functions into different domains of the protein. Striking examples are enzymes, which in addition to their catalytic function are involved in fully unrelated processes such as autophagy, protein transport or DNA maintenance. In this contribution we present an overview of our current knowledge of moonlighting proteins and discuss the significant implications for biomedical and fundamental research. 2010 Elsevier B.V. All rights reserved.

  7. Temporal form in interaction design

    DEFF Research Database (Denmark)

    Vallgårda, Anna; Winther, Morten Trøstrup; Mørch, Nina

    2015-01-01

    Interaction design is distinguished from most other design disciplines through its temporal form. Temporal form is the computational structure that enables and demands a temporal expression in the resulting design. Temporal form is what enables poetry. In music, temporal form is the composition o...

  8. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein.

    Science.gov (United States)

    Shukla, Abhinav A; Gupta, Priyanka; Han, Xuejun

    2007-11-09

    Protein A chromatography has come to be widely adopted for large-scale purification of monoclonal antibodies and Fc fusion proteins. The low pH conditions required for Protein A elution can often lead to aggregation issues for these products. A concerted study of the kinetics of aggregate formation and their relation to chromatography on Protein A media has been lacking. This paper provides a framework to describe aggregation kinetics for an Fc fusion protein that was highly susceptible to aggregate formation under low pH conditions. In contrast to what is usually expected to be a higher order reaction, first order aggregation kinetics were observed for this protein over a wide range of conditions. A comparison of the rate constants of aggregation forms an effective means of comparing various stabilizing additives to the elution buffer with one another. Inclusion of urea in the elution buffer at moderate concentrations (Protein A column were both found to be effective solutions to the aggregation issue. Elution from the Protein A resin was found to increase the aggregation rate constants over and above what would be expected from exposure to low pH conditions in solution alone. This demonstrates that Protein A-Fc interactions can destabilize product structure and increase the tendency to aggregate. The results presented here are anticipated to assist the development of Protein A process conditions for products that are prone to form high molecular weight aggregates during column elution.

  9. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  10. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat de Protéine de Petit-Lait, Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas ...

  11. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  12. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  13. Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

    Science.gov (United States)

    Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

    2013-01-01

    We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

  14. The Analysis of Forming Forces in Single Point Incremental Forming

    Directory of Open Access Journals (Sweden)

    Koh Kyung Hee

    2016-01-01

    Full Text Available Incremental forming is a process to produce sheet metal parts in quick. Because there is no need for dedicated dies and molds, this process is less cost and time spent. The purpose of this study is to investigate forming forces in single point incremental forming. Producing a cone frustum of aluminum is tested for forming forces. A dynamometer is used to collect forming forces and analyze them. These forces are compared with cutting forces upon producing same geometrical shapes of experimental parts. The forming forces in Z direction are 40 times larger than the machining forces. A spindle and its axis of a forming machine should be designed enough to withstand the forming forces.

  15. DENDRIMER CONJUGATES FOR SELECTIVE OF PROTEIN AGGREGATES

    DEFF Research Database (Denmark)

    2004-01-01

    Dendrimer conjugates are presented, which are formed between a dendrimer and a protein solubilising substance. Such dendrimer conjugates are effective in the treatment of protein aggregate-related diseases (e.g. prion-related diseases). The protein solubilising substance and the dendrimer together...

  16. Receptors, G proteins, and their interactions

    NARCIS (Netherlands)

    Hollmann, Markus W.; Strumper, Danja; Herroeder, Susanne; Durieux, Marcel E.

    2005-01-01

    Membrane receptors coupling to intracellular G proteins (G protein-coupled receptors) form one of the major classes of membrane signaling proteins. They are of great importance to the practice of anesthesiology because they are involved in many systems of relevance to the specialty (cardiovascular

  17. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

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

  18. Rheology and structure of milk protein gels

    NARCIS (Netherlands)

    Vliet, van T.; Lakemond, C.M.M.; Visschers, R.W.

    2004-01-01

    Recent studies on gel formation and rheology of milk gels are reviewed. A distinction is made between gels formed by aggregated casein, gels of `pure` whey proteins and gels in which both casein and whey proteins contribute to their properties. For casein' whey protein mixtures, it has been shown

  19. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  20. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    proteins. Together these components co‐operate in cargo‐selection as well as forming, loading and releasing budding vesicles from specific regions on the membrane surface of the ER. Coat components furthermore convey vesicle targeting towards the Golgi. However, not much is known about the mechanisms...... that regulate the COPII assembly at the vesicle bud site. This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP). The aim of the project was to characterize p125A function by dissecting two main domains...... in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization. We first show, that the DDHD...

  1. Advances in metal forming expert system for metal forming

    CERN Document Server

    Hingole, Rahulkumar Shivajirao

    2015-01-01

    This comprehensive book offers a clear account of the theory and applications of advanced metal forming. It provides a detailed discussion of specific forming processes, such as deep drawing, rolling, bending extrusion and stamping. The author highlights recent developments of metal forming technologies and explains sound, new and powerful expert system techniques for solving advanced engineering problems in metal forming. In addition, the basics of expert systems, their importance and applications to metal forming processes, computer-aided analysis of metalworking processes, formability analysis, mathematical modeling and case studies of individual processes are presented.

  2. Roles of residues in the interface of transient protein-protein complexes before complexation

    Science.gov (United States)

    Swapna, Lakshmipuram S.; Bhaskara, Ramachandra M.; Sharma, Jyoti; Srinivasan, Narayanaswamy

    2012-01-01

    Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for ∼65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition. PMID:22451863

  3. Tectonics: The meaning of form

    DEFF Research Database (Denmark)

    Christiansen, Karl; Brandt, Per Aage

    Tectonics – The meaning of form deals with one of the core topics of architecture: the relationship between form and content. In the world of architecture, form is not only made from brick, glass and wood. Form means something. When a material is processed with sufficient technical skill and insi......Tectonics – The meaning of form deals with one of the core topics of architecture: the relationship between form and content. In the world of architecture, form is not only made from brick, glass and wood. Form means something. When a material is processed with sufficient technical skill...... perspectives. You can read the chapters in any order you like – from the beginning, end or the middle. There is no correct order. The project is methodologically inductive: the more essays you read, the broader your knowledge of tectonics get....

  4. Protein deamidation

    OpenAIRE

    Robinson, Noah E.

    2002-01-01

    A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically ...

  5. Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy. A comparative study with C-terminal (109-141)- and N-terminal (1-86)-region-specific PTHrP radioassay

    Energy Technology Data Exchange (ETDEWEB)

    Suehiro, Mitsuko; Murakami, Minoru; Fukuchi, Minoru (Hyogo Coll. of Medicine, Nishinomiya (Japan))

    1994-11-01

    We evaluated the circulating forms of immunoreactive parathyroid hormone-related protein(PTHrP) in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1 : 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) humoral hypercalcemia malignancy(HHM) patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1 : 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r=0.86). The values also showed a close correlation with serum Ca; r=0.75 for C-RAS and r=0.81 for N-RAS. In addition, the correlation between the PTHrP reading obtained with the different RASs and serum Cr were: r=0.42 with C-RAS and r=0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP (109-141). In contrast, circulating concentrations of the PTHrP (1-86) fragment are very low, but detection of the PTHrP (1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PHPrP (109-141) fragment with C-RAS. (author).

  6. Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy. A comparative study with C-terminal (109-141)- and N-terminal (1-86)-region-specific PTHrP radioassay

    International Nuclear Information System (INIS)

    Suehiro, Mitsuko; Murakami, Minoru; Fukuchi, Minoru

    1994-01-01

    We evaluated the circulating forms of immunoreactive parathyroid hormone-related protein(PTHrP) in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1 : 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) humoral hypercalcemia malignancy(HHM) patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1 : 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r=0.86). The values also showed a close correlation with serum Ca; r=0.75 for C-RAS and r=0.81 for N-RAS. In addition, the correlation between the PTHrP reading obtained with the different RASs and serum Cr were: r=0.42 with C-RAS and r=0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP (109-141). In contrast, circulating concentrations of the PTHrP (1-86) fragment are very low, but detection of the PTHrP (1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PHPrP (109-141) fragment with C-RAS. (author)

  7. Proteomic screening for amyloid proteins.

    Directory of Open Access Journals (Sweden)

    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  8. Protein oxidation in aging and the removal of oxidized proteins.

    Science.gov (United States)

    Höhn, Annika; König, Jeannette; Grune, Tilman

    2013-10-30

    Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Rational design of fiber forming supramolecular structures

    Science.gov (United States)

    Wang, Benjamin K; Kanahara, Satoko M

    2016-01-01

    Recent strides in the development of multifunctional synthetic biomimetic materials through the self-assembly of multi-domain peptides and proteins over the past decade have been realized. Such engineered systems have wide-ranging application in bioengineering and medicine. This review focuses on fundamental fiber forming α-helical coiled-coil peptides, peptide amphiphiles, and amyloid-based self-assembling peptides; followed by higher order collagen- and elastin-mimetic peptides with an emphasis on chemical / biological characterization and biomimicry. PMID:27022140

  10. Adaptive Municipal e-forms

    NARCIS (Netherlands)

    Kuiper, P.M.; van Dijk, Elisabeth M.A.G.; Boerma, A.K.; Weibelzahl, S.; Cristea, A.

    2006-01-01

    Adaptation of electronic forms seems to be a step forward to reduce the burden for people who fill in forms. Municipalities more and more offer eforms online that can be used to request a municipal product or service. To create adaptive e-forms that satisfy the need of end-users, involvement of

  11. Protein Adsorption in Three Dimensions

    Science.gov (United States)

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  12. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Bernhard [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany); Lecker, Laura S. M.; Zoltner, Martin [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Jaenicke, Elmar [Johannes Gutenberg-Universität, Jakob Welder Weg 26, 55128 Mainz (Germany); Schnell, Robert [Karolinska Institutet, 17 177 Stockholm (Sweden); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Brenk, Ruth, E-mail: w.n.hunter@dundee.ac.uk [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany)

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  13. 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

    . The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  14. A Stevedore's protein knot.

    Directory of Open Access Journals (Sweden)

    Daniel Bölinger

    2010-04-01

    Full Text Available Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered alpha-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip.

  15. Protein Crystallizability.

    Science.gov (United States)

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  16. Cry protein crystals: a novel platform for protein delivery.

    Science.gov (United States)

    Nair, Manoj S; Lee, Marianne M; Bonnegarde-Bernard, Astrid; Wallace, Julie A; Dean, Donald H; Ostrowski, Michael C; Burry, Richard W; Boyaka, Prosper N; Chan, Michael K

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

  17. Cry protein crystals: a novel platform for protein delivery.

    Directory of Open Access Journals (Sweden)

    Manoj S Nair

    Full Text Available Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

  18. Cry Protein Crystals: A Novel Platform for Protein Delivery

    Science.gov (United States)

    Bonnegarde-Bernard, Astrid; Wallace, Julie A.; Dean, Donald H.; Ostrowski, Michael C.; Burry, Richard W.; Boyaka, Prosper N.; Chan, Michael K.

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues. PMID:26030844

  19. Harmony of super form factors

    Science.gov (United States)

    Brandhuber, A.; Gürdoğan, Ö.; Mooney, R.; Travaglini, G.; Yang, G.

    2011-10-01

    In this paper we continue our systematic study of form factors of half-BPS operators in mathcal{N} = 4 super Yang-Mills. In particular, we extend various on-shell techniques known for amplitudes to the case of form factors, including MHV rules, recursion relations, unitarity and dual MHV rules. As an application, we present the solution of the recursion relation for split-helicity form factors. We then consider form factors of the stress-tensor multiplet operator and of its chiral truncation, and write down supersymmetric Ward identities using chiral as well as non-chiral superspace formalisms. This allows us to obtain compact formulae for families of form factors, such as the maximally non-MHV case. Finally we generalise dual MHV rules in dual momentum space to form factors.

  20. FormTracer. A mathematica tracing package using FORM

    Science.gov (United States)

    Cyrol, Anton K.; Mitter, Mario; Strodthoff, Nils

    2017-10-01

    We present FormTracer, a high-performance, general purpose, easy-to-use Mathematica tracing package which uses FORM. It supports arbitrary space and spinor dimensions as well as an arbitrary number of simple compact Lie groups. While keeping the usability of the Mathematica interface, it relies on the efficiency of FORM. An additional performance gain is achieved by a decomposition algorithm that avoids redundant traces in the product tensors spaces. FormTracer supports a wide range of syntaxes which endows it with a high flexibility. Mathematica notebooks that automatically install the package and guide the user through performing standard traces in space-time, spinor and gauge-group spaces are provided. Program Files doi:http://dx.doi.org/10.17632/7rd29h4p3m.1 Licensing provisions: GPLv3 Programming language: Mathematica and FORM Nature of problem: Efficiently compute traces of large expressions Solution method: The expression to be traced is decomposed into its subspaces by a recursive Mathematica expansion algorithm. The result is subsequently translated to a FORM script that takes the traces. After FORM is executed, the final result is either imported into Mathematica or exported as optimized C/C++/Fortran code. Unusual features: The outstanding features of FormTracer are the simple interface, the capability to efficiently handle an arbitrary number of Lie groups in addition to Dirac and Lorentz tensors, and a customizable input-syntax.