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Sample records for protein molecules populate

  1. Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

    Science.gov (United States)

    Langdon, Blake B; Mirhossaini, Roya B; Mabry, Joshua N; Sriram, Indira; Lajmi, Ajay; Zhang, Yanxia; Rojas, Orlando J; Schwartz, Daniel K

    2015-02-18

    Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations

  2. Hierarchical organization in aggregates of protein molecules

    DEFF Research Database (Denmark)

    Bohr, Henrik; Kyhle, Anders; Sørensen, Alexis Hammer

    1997-01-01

    of the solution and the density of protein are varied shows the existence of specific growth processes resulting in different branch-like structures. The resulting structures are strongly influenced by the shape of each protein molecule. Lysozyme and ribonuclease are found to form spherical structures...

  3. Protein Scaffolding for Small Molecule Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Baker, David [Univ. of Washington, Seattle, WA (United States)

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  4. Aging-From molecules to populations

    DEFF Research Database (Denmark)

    Sander, Miriam; Avlund, Kirsten; Lauritzen, Martin

    2008-01-01

    of human aging. To foster interactions and collaboration between diverse scientists interested in the biochemical, physiological, epidemiological and psychosocial aspects of aging, The University of Copenhagen Faculty of Health Sciences recently organized and co-sponsored a workshop entitled Aging......-From Molecules to Populations. The following questions about human aging were discussed at the workshop: What is the limit of human life expectancy? What are the key indicators of human aging? What are the key drivers of human aging? Which genes have the greatest impact on human aging? How similar is aging...

  5. Optical probing of single fluorescent molecules and proteins

    NARCIS (Netherlands)

    Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

    2001-01-01

    Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

  6. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  7. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

  8. Simulation of diffusion time of small molecules in protein crystals.

    Science.gov (United States)

    Geremia, Silvano; Campagnolo, Mara; Demitri, Nicola; Johnson, Louise N

    2006-03-01

    A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.

  9. Small molecule annotation for the Protein Data Bank.

    Science.gov (United States)

    Sen, Sanchayita; Young, Jasmine; Berrisford, John M; Chen, Minyu; Conroy, Matthew J; Dutta, Shuchismita; Di Costanzo, Luigi; Gao, Guanghua; Ghosh, Sutapa; Hudson, Brian P; Igarashi, Reiko; Kengaku, Yumiko; Liang, Yuhe; Peisach, Ezra; Persikova, Irina; Mukhopadhyay, Abhik; Narayanan, Buvaneswari Coimbatore; Sahni, Gaurav; Sato, Junko; Sekharan, Monica; Shao, Chenghua; Tan, Lihua; Zhuravleva, Marina A

    2014-01-01

    The Protein Data Bank (PDB) is the single global repository for three-dimensional structures of biological macromolecules and their complexes, and its more than 100,000 structures contain more than 20,000 distinct ligands or small molecules bound to proteins and nucleic acids. Information about these small molecules and their interactions with proteins and nucleic acids is crucial for our understanding of biochemical processes and vital for structure-based drug design. Small molecules present in a deposited structure may be attached to a polymer or may occur as a separate, non-covalently linked ligand. During curation of a newly deposited structure by wwPDB annotation staff, each molecule is cross-referenced to the PDB Chemical Component Dictionary (CCD). If the molecule is new to the PDB, a dictionary description is created for it. The information about all small molecule components found in the PDB is distributed via the ftp archive as an external reference file. Small molecule annotation in the PDB also includes information about ligand-binding sites and about covalent and other linkages between ligands and macromolecules. During the remediation of the peptide-like antibiotics and inhibitors present in the PDB archive in 2011, it became clear that additional annotation was required for consistent representation of these molecules, which are quite often composed of several sequential subcomponents including modified amino acids and other chemical groups. The connectivity information of the modified amino acids is necessary for correct representation of these biologically interesting molecules. The combined information is made available via a new resource called the Biologically Interesting molecules Reference Dictionary, which is complementary to the CCD and is now routinely used for annotation of peptide-like antibiotics and inhibitors. © The Author(s) 2014. Published by Oxford University Press.

  10. Semisynthetic protein nanoreactor for single-molecule chemistry

    OpenAIRE

    Lee, Joongoo; Bayley, Hagan

    2015-01-01

    The modulation of ionic current flowing through an individual protein pore provides information at the single-molecule level about chemical reactions occurring within the pore. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. H...

  11. Surface Passivation for Single-molecule Protein Studies

    Science.gov (United States)

    Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-01-01

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation. PMID:24797261

  12. Statistical inference in single molecule measurements of protein adsorption

    Science.gov (United States)

    Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

    2018-02-01

    Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

  13. Reciprocal carbonyl-carbonyl interactions in small molecules and proteins.

    Science.gov (United States)

    Rahim, Abdur; Saha, Pinaki; Jha, Kunal Kumar; Sukumar, Nagamani; Sarma, Bani Kanta

    2017-07-19

    Carbonyl-carbonyl n→π* interactions where a lone pair (n) of the oxygen atom of a carbonyl group is delocalized over the π* orbital of a nearby carbonyl group have attracted a lot of attention in recent years due to their ability to affect the 3D structure of small molecules, polyesters, peptides, and proteins. In this paper, we report the discovery of a "reciprocal" carbonyl-carbonyl interaction with substantial back and forth n→π* and π→π* electron delocalization between neighboring carbonyl groups. We have carried out experimental studies, analyses of crystallographic databases and theoretical calculations to show the presence of this interaction in both small molecules and proteins. In proteins, these interactions are primarily found in polyproline II (PPII) helices. As PPII are the most abundant secondary structures in unfolded proteins, we propose that these local interactions may have implications in protein folding.Carbonyl-carbonyl π* non covalent interactions affect the structure and stability of small molecules and proteins. Here, the authors carry out experimental studies, analyses of crystallographic databases and theoretical calculations to describe an additional type of carbonyl-carbonyl interaction.

  14. Vibrational relaxation induced population inversions in laser pumped polyatomic molecules

    International Nuclear Information System (INIS)

    Shamah, I.; Flynn, G.; Columbia Univ., New York

    1981-01-01

    Conditions for population inversion in laser pumped polyatomic molecules are described. For systems which exhibit metastable vibrational population distributions, large, long lived inversions are possible even when the vibrational modes are strongly coupled by rapid collisional vibration-vibration (V-V) energy transfer. Overtone states of a hot mode are found to invert with respect to fundamental levels of a cold mode even at V-V steady state. Inversion persists for a V-T/R relaxation time. A gain of 4 m -1 for the 2ν 3 → ν 2 transition in CH 3 F (lambda approx. 15.9 μ) was found assuming a spontaneous emission lifetime of 10 s for this transition. General equations are derived which can be used to determine the magnitude of population inversion in any laser pumped, vibrationally metastable, polyatomic molecule. A discussion of factors controlling the population maxima of different vibrational states in optically pumped, V-V equilibrated metastable polyatomics is also given. (orig./WL)

  15. Single-molecule protein sequencing through fingerprinting: computational assessment

    Science.gov (United States)

    Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

    2015-10-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

  16. Single-molecule protein sequencing through fingerprinting: computational assessment

    International Nuclear Information System (INIS)

    Yao, Yao; Docter, Margreet; Van Ginkel, Jetty; Joo, Chirlmin; De Ridder, Dick

    2015-01-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences. (paper)

  17. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  18. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  19. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  20. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  1. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  2. Click strategies for single-molecule protein fluorescence.

    Science.gov (United States)

    Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

    2012-03-21

    Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

  3. Urea transporter proteins as targets for small-molecule diuretics.

    Science.gov (United States)

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

  4. Two states or not two states: Single-molecule folding studies of protein L

    Science.gov (United States)

    Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

    2018-03-01

    Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

  5. Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors.

    Directory of Open Access Journals (Sweden)

    Arnout Voet

    Full Text Available One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs. We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.

  6. Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics.

    Science.gov (United States)

    Parra, R Gonzalo; Schafer, Nicholas P; Radusky, Leandro G; Tsai, Min-Yeh; Guzovsky, A Brenda; Wolynes, Peter G; Ferreiro, Diego U

    2016-07-08

    The protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL: http://frustratometer.qb.fcen.uba.ar. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Structural studies on proton/protonation of the protein molecule

    International Nuclear Information System (INIS)

    Morimoto, Yukio; Kida, Akiko; Chatake, Toshiyuki; Yamaguchi, Hiroshi; Hosokawa, Keiichi; Murakami, Takuto; Umino, Masaaki; Tanaka, Ichiro; Hisatome, Ichiro; Yanagisawa, Yasutake; Fujiwara, Satoshi; Hidaka, Yuji; Shimamoto, Shigeru; Fujiwara, Mitsutoshi; Nakanishi, Takeyoshi

    2015-01-01

    This paper reports three studies involved in the analysis of protons and protonation at physiologically active sites in protein molecules. (1) 'Elucidation of the higher-order structure formation and activity performing mechanism of yeast proteasome.' With an aim to apply to anti-cancer drugs, this study performed the shape analysis of the total structure of 26S proteasome using small-angle X-ray scattering to clarify the complex form where controlling elements bonded to the both ends of 20S catalyst body, and analyzed the complex structure between the active sites of 20S and inhibitor (drug). (2) 'Basic study on the neutron experiment of biomolecules such as physiologically active substances derived from Natto-bacteria.' This study conducted the purification, crystallization, and X-ray analysis experiment of nattokinase; high-grade purification and solution experiment of vitamin K2 (menaquinone-7); and Z-DNA crystal structure study related to the neutron crystal analysis of DNA as another biomolecule structure study. (3) 'Functional evaluation on digestive enzymes derived from Nephila clavata.' As an Alzheimer's disease-related amyloid fibril formation model, this study carried out elucidation on the fibrosis and fiber-forming mechanism of the traction fiber of Nephila clavata, and the functional analysis of its degrading enzyme. (A.O.)

  8. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  9. Water-mediated influence of a crowded environment on internal vibrations of a protein molecule.

    Science.gov (United States)

    Kuffel, Anna; Zielkiewicz, Jan

    2016-02-14

    The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules. The presence of additional molecules influences the amplitudes of protein fluctuations. Also, a weak dynamical coupling of collective velocities of surface atoms of proteins separated by a layer of water is detected. The possible mechanisms of these phenomena are described. The cross-correlation function of the collective velocities of surface atoms of two proteins was decomposed into the Fourier series. The amplitude spectrum displays a peak at low frequencies. Also, the results of principal component analysis suggest that the close presence of an additional protein molecule influences the high-amplitude, low-frequency modes in the most prominent way. This part of the spectrum covers biologically important protein motions. The neighbor-induced changes in the inner dynamics of the protein may be connected with the changes in the velocity power spectrum of interfacial water. The additional protein molecule changes the properties of solvation water and in this way it can influence the dynamics of the second protein. It is suggested that this phenomenon may be described, at first approximation, by a damped oscillator driven by an external random force. This model was successfully applied to conformationally rigid Choristoneura fumiferana antifreeze protein molecules.

  10. A Structural Perspective on the Modulation of Protein-Protein Interactions with Small Molecules.

    Science.gov (United States)

    Demirel, Habibe Cansu; Dogan, Tunca; Tuncbag, Nurcan

    2018-05-31

    Protein-protein interactions (PPIs) are the key components in many cellular processes including signaling pathways, enzymatic reactions and epigenetic regulation. Abnormal interactions of some proteins may be pathogenic and cause various disorders including cancer and neurodegenerative diseases. Although inhibiting PPIs with small molecules is a challenging task, it gained an increasing interest because of its strong potential for drug discovery and design. The knowledge of the interface as well as the structural and chemical characteristics of the PPIs and their roles in the cellular pathways are necessary for a rational design of small molecules to modulate PPIs. In this study, we review the recent progress in the field and detail the physicochemical properties of PPIs including binding hot spots with a focus on structural methods. Then, we review recent approaches for structural prediction of PPIs. Finally, we revisit the concept of targeting PPIs in a systems biology perspective and we refer to the non-structural approaches, usually employed when the structural information is not present. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Global analysis of small molecule binding to related protein targets.

    Directory of Open Access Journals (Sweden)

    Felix A Kruger

    2012-01-01

    Full Text Available We report on the integration of pharmacological data and homology information for a large scale analysis of small molecule binding to related targets. Differences in small molecule binding have been assessed for curated pairs of human to rat orthologs and also for recently diverged human paralogs. Our analysis shows that in general, small molecule binding is conserved for pairs of human to rat orthologs. Using statistical tests, we identified a small number of cases where small molecule binding is different between human and rat, some of which had previously been reported in the literature. Knowledge of species specific pharmacology can be advantageous for drug discovery, where rats are frequently used as a model system. For human paralogs, we demonstrate a global correlation between sequence identity and the binding of small molecules with equivalent affinity. Our findings provide an initial general model relating small molecule binding and sequence divergence, containing the foundations for a general model to anticipate and predict within-target-family selectivity.

  12. Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

    Science.gov (United States)

    2017-06-26

    SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to...enhance our understanding of the interaction of proteins and surfaces. Given this objective, the specific aims of this research were to: 1) exploit the...incorporation of unnatural amino acids in proteins to introduce single-molecule probes (i.e., fluorophores for fluorescence resonance energy transfer

  13. Hot spot-based design of small-molecule inhibitors for protein-protein interactions.

    Science.gov (United States)

    Guo, Wenxing; Wisniewski, John A; Ji, Haitao

    2014-06-01

    Protein-protein interactions (PPIs) are important targets for the development of chemical probes and therapeutic agents. From the initial discovery of the existence of hot spots at PPI interfaces, it has been proposed that hot spots might provide the key for developing small-molecule PPI inhibitors. However, there has been no review on the ways in which the knowledge of hot spots can be used to achieve inhibitor design, nor critical examination of successful examples. This Digest discusses the characteristics of hot spots and the identification of druggable hot spot pockets. An analysis of four examples of hot spot-based design reveals the importance of this strategy in discovering potent and selective PPI inhibitors. A general procedure for hot spot-based design of PPI inhibitors is outlined. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. New protein involved in the replacement of cell molecules

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave

    2011-01-01

    In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed” compartme......In collaboration with colleagues from La Trobe University, Australia, scientists at Aarhus University have discovered and defined a novel enzyme involved in the replacement and renewal of cell molecules. The enzyme exerts its function within the so-called mitochondria - small “enclosed...

  15. Structural Approaches to Sequence Evolution Molecules, Networks, Populations

    CERN Document Server

    Bastolla, Ugo; Roman, H. Eduardo; Vendruscolo, Michele

    2007-01-01

    Structural requirements constrain the evolution of biological entities at all levels, from macromolecules to their networks, right up to populations of biological organisms. Classical models of molecular evolution, however, are focused at the level of the symbols - the biological sequence - rather than that of their resulting structure. Now recent advances in understanding the thermodynamics of macromolecules, the topological properties of gene networks, the organization and mutation capabilities of genomes, and the structure of populations make it possible to incorporate these key elements into a broader and deeply interdisciplinary view of molecular evolution. This book gives an account of such a new approach, through clear tutorial contributions by leading scientists specializing in the different fields involved.

  16. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.; Thomas, Ludivine; Lilley, Kathryn S.; Marondedze, Claudius

    2013-01-01

    in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through

  17. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  18. Biophysical characterization of membrane protein-small molecule interactions

    NARCIS (Netherlands)

    Chen, Dan

    2015-01-01

    Membrane proteins are account for up to two thirds of known druggable targets. Traditionally, new drugs against this class of proteins have been discovered through HTS. However, not all GPCRs are amenable to traditional screening methods. Recently, fragment-based drug discovery (FBDD) has emerged as

  19. Low dose radiation induced protein and its effect on expression of CD25 molecule in lymphocytes

    International Nuclear Information System (INIS)

    Lu Duicai; Su Liaoyuan

    2001-01-01

    Objective: To find the substantial basis for effects of low dose radiation, on development, extraction, and the biogical activity of the low-dose radiation-induced proteins, and the effects of LDR induced proteins on CD25 molecule expression of human lymphocytes. Methods: 1. Healthy Kumning male mice exposed to radiation of 226 Ra γ-rays at 5, 10 and 15 cGy respectively. The mice were killed 2 hours after exposure, the spleen cells were broken with ultrasonic energy and then ultra-centrifugalized at low temperature (4 degree C). The LDR-induced proteins were obtained in the supernatant solution. Then the changes of CD25 molecule was measured by flow cytometry (FCM) with immunofluorescence technique, which was used to reflect the effect of LDR induced proteins on CD25 molecule expression of human lymphocytes. Results: LDR induced proteins were obtained from spleen cells in mice exposed to 5-15 cGy whole body radiation. Conclusion: The expression of CD25 molecule of lymphocytes was increased significantly after use of LDR induced proteins. LDR induced proteins can enhance expression of CD25 molecule of lymphocytes slightly

  20. Tubular kidney injury molecule-1 in protein-overload nephropathy

    NARCIS (Netherlands)

    van Timmeren, Mirjan M.; Bakker, Stephan J. L.; Vaidya, Vishal S.; Bailly, Veronique; Schuurs, Theo A.; Damman, Jeffrey; Stegeman, Coen A.; Bonventre, Joseph V.; van Goor, Harry

    Kim-1, a recently discovered membrane protein, is undetectable in normal kidneys but markedly induced in proximal tubules after ischemic and toxic injury. The function of Kim-1 is unclear, but it is implicated in damage/repair processes. The Kim-1 ectodomain is cleaved by metalloproteinases and

  1. Protein folding and translocation : single-molecule investigations

    NARCIS (Netherlands)

    Leeuwen, Rudolphus Gerardus Henricus van

    2006-01-01

    This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly

  2. Single-Molecule Studies of Bacterial Protein Translocation

    NARCIS (Netherlands)

    Kedrov, Alexej; Kusters, Ilja; Driessen, Arnold J. M.

    2013-01-01

    In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of

  3. Towards single-molecule observation of protein synthesis

    International Nuclear Information System (INIS)

    Dulin, David; Le Gall, Antoine; Bouyer, Philippe; Perronet, Karen; Westbrook, Nathalie; Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2009-01-01

    The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, a study at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the development of an advanced surface chemistry to attach an active ribosome to the microscope coverslip and follow the amino-acid incorporation by fluorescence microscopy. The ribosome is labeled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. It should then be possible to observe the protein synthesis in real time as the labeled amino acids are incorporated into the polypeptide chain. (Author)

  4. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

    NARCIS (Netherlands)

    van Mameren, J.; Peterman, E.J.G.; Wuite, G.J.L.

    2008-01-01

    Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of

  5. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

    Science.gov (United States)

    Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

    2013-06-14

    Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

  6. Difficulties in applying pure Kohn-Sham density functional theory electronic structure methods to protein molecules

    Science.gov (United States)

    Rudberg, Elias

    2012-02-01

    Self-consistency-based Kohn-Sham density functional theory (KS-DFT) electronic structure calculations with Gaussian basis sets are reported for a set of 17 protein-like molecules with geometries obtained from the Protein Data Bank. It is found that in many cases such calculations do not converge due to vanishing HOMO-LUMO gaps. A sequence of polyproline I helix molecules is also studied and it is found that self-consistency calculations using pure functionals fail to converge for helices longer than six proline units. Since the computed gap is strongly correlated to the fraction of Hartree-Fock exchange, test calculations using both pure and hybrid density functionals are reported. The tested methods include the pure functionals BLYP, PBE and LDA, as well as Hartree-Fock and the hybrid functionals BHandHLYP, B3LYP and PBE0. The effect of including solvent molecules in the calculations is studied, and it is found that the inclusion of explicit solvent molecules around the protein fragment in many cases gives a larger gap, but that convergence problems due to vanishing gaps still occur in calculations with pure functionals. In order to achieve converged results, some modeling of the charge distribution of solvent water molecules outside the electronic structure calculation is needed. Representing solvent water molecules by a simple point charge distribution is found to give non-vanishing HOMO-LUMO gaps for the tested protein-like systems also for pure functionals.

  7. Difficulties in applying pure Kohn-Sham density functional theory electronic structure methods to protein molecules

    International Nuclear Information System (INIS)

    Rudberg, Elias

    2012-01-01

    Self-consistency-based Kohn-Sham density functional theory (KS-DFT) electronic structure calculations with Gaussian basis sets are reported for a set of 17 protein-like molecules with geometries obtained from the Protein Data Bank. It is found that in many cases such calculations do not converge due to vanishing HOMO-LUMO gaps. A sequence of polyproline I helix molecules is also studied and it is found that self-consistency calculations using pure functionals fail to converge for helices longer than six proline units. Since the computed gap is strongly correlated to the fraction of Hartree-Fock exchange, test calculations using both pure and hybrid density functionals are reported. The tested methods include the pure functionals BLYP, PBE and LDA, as well as Hartree-Fock and the hybrid functionals BHandHLYP, B3LYP and PBE0. The effect of including solvent molecules in the calculations is studied, and it is found that the inclusion of explicit solvent molecules around the protein fragment in many cases gives a larger gap, but that convergence problems due to vanishing gaps still occur in calculations with pure functionals. In order to achieve converged results, some modeling of the charge distribution of solvent water molecules outside the electronic structure calculation is needed. Representing solvent water molecules by a simple point charge distribution is found to give non-vanishing HOMO-LUMO gaps for the tested protein-like systems also for pure functionals. (fast track communication)

  8. Rapid and accurate prediction and scoring of water molecules in protein binding sites.

    Directory of Open Access Journals (Sweden)

    Gregory A Ross

    Full Text Available Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity.

  9. Protein-based nanostructures as carriers for photo-physically active molecules in biosystems

    OpenAIRE

    Delcanale, Pietro

    2017-01-01

    In nature, many proteins function as carriers, being able to bind, transport and possibly release a ligand within a biological system. Protein-based carriers are interesting systems for drug delivery, with the remarkable advantage of being water-soluble and, as inherent components of biosystems, highly bio-compatible. This work focuses on the use of protein-based carriers for the delivery of hydrophobic photo-physically active molecules, whose structure and chemical properties lead to spontan...

  10. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Directory of Open Access Journals (Sweden)

    Marijn T J van Loenhout

    Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  11. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  12. Steering population transfer of the Na2 molecule by an ultrashort pulse train

    Science.gov (United States)

    Niu, Dong-Hua; Wang, Shuo; Zhan, Wei-Shen; Tao, Hong-Cai; Wang, Si-Qi

    2018-05-01

    We theoretically investigate the complete population transfer among quantum states of the Na2 molecule using ultrashort pulse trains using the time-dependent wave packet method. The population accumulation of the target state can be steered by controlling the laser parameters, such as the variable pulse pairs, the different pulse widths, the time delays and the repetition period between two contiguous pulses; in particular, the pulse pairs and the pulse widths have a great effect on the population transfer. The calculations show that the ultrashort pulse train is a feasible solution, which can steer the population transfer from the initial state to the target state efficiently with lower peak intensities.

  13. ASDB: a resource for probing protein functions with small molecules.

    Science.gov (United States)

    Liu, Zhihong; Ding, Peng; Yan, Xin; Zheng, Minghao; Zhou, Huihao; Xu, Yuehua; Du, Yunfei; Gu, Qiong; Xu, Jun

    2016-06-01

    : Identifying chemical probes or seeking scaffolds for a specific biological target is important for protein function studies. Therefore, we create the Annotated Scaffold Database (ASDB), a computer-readable and systematic target-annotated scaffold database, to serve such needs. The scaffolds in ASDB were derived from public databases including ChEMBL, DrugBank and TCMSP, with a scaffold-based classification approach. Each scaffold was assigned with an InChIKey as its unique identifier, energy-minimized 3D conformations, and other calculated properties. A scaffold is also associated with drugs, natural products, drug targets and medical indications. The database can be retrieved through text or structure query tools. ASDB collects 333 601 scaffolds, which are associated with 4368 targets. The scaffolds consist of 3032 scaffolds derived from drugs and 5163 scaffolds derived from natural products. For given scaffolds, scaffold-target networks can be generated from the database to demonstrate the relations of scaffolds and targets. ASDB is freely available at http://www.rcdd.org.cn/asdb/with the major web browsers. junxu@biochemomes.com or xujun9@mail.sysu.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Prediction of small molecule binding property of protein domains with Bayesian classifiers based on Markov chains.

    Science.gov (United States)

    Bulashevska, Alla; Stein, Martin; Jackson, David; Eils, Roland

    2009-12-01

    Accurate computational methods that can help to predict biological function of a protein from its sequence are of great interest to research biologists and pharmaceutical companies. One approach to assume the function of proteins is to predict the interactions between proteins and other molecules. In this work, we propose a machine learning method that uses a primary sequence of a domain to predict its propensity for interaction with small molecules. By curating the Pfam database with respect to the small molecule binding ability of its component domains, we have constructed a dataset of small molecule binding and non-binding domains. This dataset was then used as training set to learn a Bayesian classifier, which should distinguish members of each class. The domain sequences of both classes are modelled with Markov chains. In a Jack-knife test, our classification procedure achieved the predictive accuracies of 77.2% and 66.7% for binding and non-binding classes respectively. We demonstrate the applicability of our classifier by using it to identify previously unknown small molecule binding domains. Our predictions are available as supplementary material and can provide very useful information to drug discovery specialists. Given the ubiquitous and essential role small molecules play in biological processes, our method is important for identifying pharmaceutically relevant components of complete proteomes. The software is available from the author upon request.

  15. Multilayer Choline Phosphate Molecule Modified Surface with Enhanced Cell Adhesion but Resistance to Protein Adsorption.

    Science.gov (United States)

    Chen, Xingyu; Yang, Ming; Liu, Botao; Li, Zhiqiang; Tan, Hong; Li, Jianshu

    2017-08-22

    Choline phosphate (CP), which is a new zwitterionic molecule, and has the reverse order of phosphate choline (PC) and could bind to the cell membrane though the unique CP-PC interaction. Here we modified a glass surface with multilayer CP molecules using surface-initiated atom-transfer radical polymerization (SI-ATRP) and the ring-opening method. Polymeric brushes of (dimethylamino)ethyl methacrylate (DMAEMA) were synthesized by SI-ATRP from the glass surface. Then the grafted PDMAEMA brushes were used to introduce CP groups to fabricate the multilayer CP molecule modified surface. The protein adsorption experiment and cell culture test were used to evaluate the biocompatibility of the modified surfaces by using human umbilical veinendothelial cells (HUVECs). The protein adsorption results demonstrated that the multilayer CP molecule decorated surface could prevent the adsorption of fibrinogen and serum protein. The adhesion and proliferation of cells were improved significantly on the multilayer CP molecule modified surface. Therefore, the biocompatibility of the material surface could be improved by the modified multilayer CP molecule, which exhibits great potential for biomedical applications, e.g., scaffolds in tissue engineering.

  16. Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle.

    Directory of Open Access Journals (Sweden)

    Ali Akgul

    Full Text Available Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis. L. monocytogenes lapB gene encodes a cell wall surface anchor protein, and mutation of this gene causes Listeria attenuation in mice. In this work, the potential role of Listeria LapB protein in catfish fillet attachment was investigated. To achieve this, boron-based small molecules designed to interfere with the active site of the L. monocytogenes LapB protein were developed, and their ability to prevent L. monocytogenes attachment to fish fillet was tested. Results indicated that seven out of nine different small molecules were effective in reducing the Listeria attachment to catfish fillets. Of these, three small molecules (SM3, SM5, and SM7 were highly effective in blocking Listeria attachment to catfish fillets. This study suggests an alternative strategy for reduction of L. monocytogenes contamination in fresh and frozen fish products.

  17. A dual small-molecule rheostat for precise control of protein concentration in Mammalian cells.

    Science.gov (United States)

    Lin, Yu Hsuan; Pratt, Matthew R

    2014-04-14

    One of the most successful strategies for controlling protein concentrations in living cells relies on protein destabilization domains (DD). Under normal conditions, a DD will be rapidly degraded by the proteasome. However, the same DD can be stabilized or "shielded" in a stoichiometric complex with a small molecule, enabling dose-dependent control of its concentration. This process has been exploited by several labs to post-translationally control the expression levels of proteins in vitro as well as in vivo, although the previous technologies resulted in permanent fusion of the protein of interest to the DD, which can affect biological activity and complicate results. We previously reported a complementary strategy, termed traceless shielding (TShld), in which the protein of interest is released in its native form. Here, we describe an optimized protein concentration control system, TTShld, which retains the traceless features of TShld but utilizes two tiers of small molecule control to set protein concentrations in living cells. These experiments provide the first protein concentration control system that results in both a wide range of protein concentrations and proteins free from engineered fusion constructs. The TTShld system has a greatly improved dynamic range compared to our previously reported system, and the traceless feature is attractive for elucidation of the consequences of protein concentration in cell biology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    Energy Technology Data Exchange (ETDEWEB)

    Kmetko, Jan [Kenyon College, Gambier, OH 43022 (United States); Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, NY 14853 (United States); Kenyon College, Gambier, OH 43022 (United States)

    2011-10-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

  19. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    International Nuclear Information System (INIS)

    Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

    2011-01-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions

  20. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    Science.gov (United States)

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  1. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    Science.gov (United States)

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  2. Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Huijun Xue

    2017-10-01

    Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

  3. Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

    NARCIS (Netherlands)

    van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

    2001-01-01

    OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte

  4. Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

    Science.gov (United States)

    Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

    2005-07-01

    We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

  5. Quantum atom-heteronuclear molecule dark state: Role of population imbalance

    International Nuclear Information System (INIS)

    Jing Hui; Cui Shuai

    2010-01-01

    Recently, the finite-number effect of initial atoms in coherent atom-molecule conversion was investigated by Zhao et al. [Phys. Rev. Lett. 101, 010401 (2008)]. Here, by extending to the atom-heteronuclear molecule dark state, we find that the initial populations imbalance of the atoms plays a significant role in quantum conversion rate and adiabatic fidelity. In particular, even for the finite total number of imbalanced two-species atoms, the mean-field conversion rate, contrary to the general belief, still can be remarkably close to the exact quantum results.

  6. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  7. Influence of Electrostatics on Small Molecule Flux through a Protein Nanoreactor.

    Science.gov (United States)

    Glasgow, Jeff E; Asensio, Michael A; Jakobson, Christopher M; Francis, Matthew B; Tullman-Ercek, Danielle

    2015-09-18

    Nature uses protein compartmentalization to great effect for control over enzymatic pathways, and the strategy has great promise for synthetic biology. In particular, encapsulation in nanometer-sized containers to create nanoreactors has the potential to elicit interesting, unexplored effects resulting from deviations from well-understood bulk processes. Self-assembled protein shells for encapsulation are especially desirable for their uniform structures and ease of perturbation through genetic mutation. Here, we use the MS2 capsid, a well-defined porous 27 nm protein shell, as an enzymatic nanoreactor to explore pore-structure effects on substrate and product flux during the catalyzed reaction. Our results suggest that the shell can influence the enzymatic reaction based on charge repulsion between small molecules and point mutations around the pore structure. These findings also lend support to the hypothesis that protein compartments modulate the transport of small molecules and thus influence metabolic reactions and catalysis in vitro.

  8. Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

    Science.gov (United States)

    Meng Zhang; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

    2014-01-01

    High throughput screening of protein-small molecule binding interactions using label-free optical biosensors is challenging, as the detected signals are often similar in magnitude to experimental noise. Here, we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with sub-picometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets with binding affinities or inhibition constants in the sub-nanomolar to low micromolar range. The demonstrated ability to perform detection in the presence of several interfering compounds opens the potential for increasing the throughput of the approach. As an example application, we performed a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II (CA II), in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

  9. Single Molecule Effects of Osteogenesis Imperfecta Mutations in Tropocollagen Protein Domains

    Science.gov (United States)

    2008-12-02

    Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains Alfonso Gautieri,1,2 Simone Vesentini,2 Alberto...2008 proteinscience.org Abstract: Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe...diagnosis and treatment, an effort referred to as materiomics. Keywords: steered molecular dynamics; osteogenesis imperfecta ; Young’s modulus; collagen

  10. Insights into the role of protein molecule size and structure on interfacial properties using designed sequences

    Science.gov (United States)

    Dwyer, Mirjana Dimitrijev; He, Lizhong; James, Michael; Nelson, Andrew; Middelberg, Anton P. J.

    2013-01-01

    Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4). Interfacial tension measurements suggested that the size and bulk structuring of the larger molecule led to much slower adsorption kinetics. Neutron reflectometry at equilibrium revealed that both molecules adsorbed as a monolayer to the air–water interface (indicating unfolding of DAMP4 to give a chain of four connected DAMP1 molecules), with a concentration ratio equal to that in the bulk. This suggests the overall free energy of adsorption is equal despite differences in size and bulk structure. At small interfacial extensional strains, only molecule packing influenced the stress response. At larger strains, the effect of size became apparent, with DAMP4 registering a higher stress response and interfacial elasticity. When both components were present at the interface, most stress-dissipating movement was achieved by DAMP1. This work thus provides insights into the role of proteins' molecular size and structure on their interfacial properties, and the designed sequences introduced here can serve as effective tools for interfacial studies of proteins and polymers. PMID:23303222

  11. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  12. The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein.

    Directory of Open Access Journals (Sweden)

    Gabriele Giachin

    Full Text Available Humic substances (HS are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of "salting-out" whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of "prion ecology". These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

  13. Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach.

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    2011-03-01

    Full Text Available Ribosome-inactivating proteins (RIPs are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL, thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2, produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2 from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

  14. THE CHEMISTRY OF POPULATION III SUPERNOVA EJECTA. I. FORMATION OF MOLECULES IN THE EARLY UNIVERSE

    International Nuclear Information System (INIS)

    Cherchneff, Isabelle; Dwek, Eli

    2009-01-01

    We study the formation and destruction of molecules in the ejecta of Population III supernovae (SNe) using a chemical kinetic approach to follow the evolution of molecular abundances from day 100 to day 1000 after explosion. The chemical species included in the study range from simple diatomic molecules to more complex dust precursor species. All relevant molecule formation and destruction processes that are unique to the SN environment are considered. Our work focuses on zero-metallicity progenitors with masses of 20, 170, and 270 M sun , and we study the effect of different levels of heavy element mixing and the inward diffusion of hydrogen and helium on the ejecta chemistry. We show that the ejecta chemistry does not reach a steady state within the relevant timespan (∼3 yr) for molecule formation, thus invalidating previous results relying on this assumption. The primary species formed in the harsh SN environment are O 2 , CO, SiS, and SO. The SiO, formed as early as 200 days after explosion, is rapidly depleted by the formation of silica molecular precursors in the ejecta. The rapid conversion of CO to C 2 and its thermal fractionation at temperatures above 5000 K allow for the formation of carbon chains in the oxygen-rich zone of the unmixed models, providing an important pathway for the formation of carbon dust in hot environments where the C/O ratio is less than 1. We show that the fully mixed ejecta of a 170 M sun progenitor synthesizes 11.3 M sun of molecules, whereas 20 M sun and 270 M sun progenitors produce 0.78 M sun and 3.2 M sun of molecules, respectively. The admixing of 10% of hydrogen into the fully mixed ejecta of the 170 M sun progenitor increases its molecular yield to ∼47 M sun . The unmixed ejecta of a 170 M sun progenitor SN without hydrogen penetration synthesizes ∼37 M sun of molecules, whereas its 20 M sun counterpart produces ∼1.2 M sun . This smaller efficiency at forming molecules is due to the large fraction of He + in the

  15. Micrometer sized immobilization of protein molecules onto quartz, silicium and gold.

    Science.gov (United States)

    Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Klitgaard, Søren; Duroux, Meg Crookshanks

    2006-02-01

    We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.

  16. Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

    Science.gov (United States)

    Zheng, Haoping

    2003-04-01

    The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with unchanged calculation precision. So the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule becomes a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the Ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), are presented. The reactive sites of the inhibitors are determined and explained. The precision of structure determination of inhibitors are tested theoretically.

  17. Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

    OpenAIRE

    Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

    2014-01-01

    Homologous recombination is a conserved pathway for repairing double?stranded breaks, which are processed to yield single?stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single?molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA?ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 b...

  18. Immobilization of small molecules and proteins by radio-derivatized polystyrene

    International Nuclear Information System (INIS)

    Varga, J.M.; Fritsch, P.

    1990-01-01

    When molded polystyrene (PS) products (e.g., microtiter plates) or latex particles are irradiated with high-energy (1-10 Mrads) gamma rays in the presence of nonpolymerizable small molecules such as aromatic amines, some of these molecules incorporate into PS, which leads to the formation of radio-derivatized PS (RDPS). Two classes of RDPS can be identified regarding their ability for immobilization of biologically important molecules: (1) reactive RDPS that are able to form covalent bonds with molecules such as proteins without the help of cross-linkers, and (2) functionalized RDPS that can be used for the immobilization of molecules with activators (e.g., carbodiimides) or cross-linkers. The method can be used for the production of low-noise supports for binding assays. Most of the RDPS can be produced without impairment of the optical quality of PS, making derivatized microtiter plates suitable for colorimetric assays. The principle can be applied for the preparation of affinity sorbents, e.g., for high-performance affinity chromatography and for the immobilization of enzymes using latex PS particles

  19. Multiple molecule effects on the cooperativity of protein folding transitions in simulations

    Science.gov (United States)

    Lewis, Jacob I.; Moss, Devin J.; Knotts, Thomas A.

    2012-06-01

    Though molecular simulation of proteins has made notable contributions to the study of protein folding and kinetics, disagreement between simulation and experiment still exists. One of the criticisms levied against simulation is its failure to reproduce cooperative protein folding transitions. This weakness has been attributed to many factors such as a lack of polarizability and adequate capturing of solvent effects. This work, however, investigates how increasing the number of proteins simulated simultaneously can affect the cooperativity of folding transitions — a topic that has received little attention previously. Two proteins are studied in this work: phage T4 lysozyme (Protein Data Bank (PDB) ID: 7LZM) and phage 434 repressor (PDB ID: 1R69). The results show that increasing the number of proteins molecules simulated simultaneously leads to an increase in the macroscopic cooperativity for transitions that are inherently cooperative on the molecular level but has little effect on the cooperativity of other transitions. Taken as a whole, the results identify one area of consideration to improving simulations of protein folding.

  20. Influence of the water molecules near surface of viral protein on virus activation process

    Energy Technology Data Exchange (ETDEWEB)

    O, Shepelenko S; S, Salnikov A; V, Rak S; P, Goncharova E; B, Ryzhikov A, E-mail: shep@vector.nsc.r, E-mail: shep@ngs.r [Federal State Research Institution State Research Center of Virology and Biotechnology VECTOR of the Federal Service for Surveillance in Consumer Rights Protection and Human Well-being (FSRI SRC VB VECTOR) Koltsovo, Novosibirsk Region (Russian Federation)

    2009-06-01

    The infection of a cell with influenza virus comprises the stages of receptor binding to the cell membrane, endocytosis of virus particle, and fusion of the virus envelope and cell endosome membrane, which is determined by the conformational changes in hemagglutinin, a virus envelope protein, caused by pH decrease within the endosome. The pH value that induces conformation rearrangements of hemagglutinin molecule considerably varies for different influenza virus strains, first and foremost, due to the differences in amino acid structure of the corresponding proteins. The main goal of this study was to construct a model making it possible to assess the critical pH value characterizing the fusogenic activity of influenza virus hemagglutinin from the data on hemagglutinin structure and experimental verification of this model. Under this model, we assume that when the electrostatic force between interacting hemagglutinin molecules in the virus envelop exceeds a certain value, the hemagglutinin HA1 subunits are arranged so that they form a cavity sufficient for penetration of water molecules. This event leads to an irreversible hydration of the inner fragments of hemagglutinin molecule in a trimer and to the completion of conformational changes. The geometry of electrostatic field in hemagglutinin trimer was calculated taking into account the polarization effects near the interface of two dielectrics, aqueous medium and protein macromolecule. The critical pH values for the conformational changes in hemagglutinin were measured by the erythrocyte hemolysis induced by influenza virus particles when decreasing pH. The critical pH value conditionally separating the pH range into the regions with and without the conformational changes was calculated for several influenza virus H1N1 and H3N2 strains based on the data on the amino acid structure of the corresponding hemagglutinin molecules. Comparison of the theoretical and experimental values of critical pH values for

  1. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

    Science.gov (United States)

    Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

    2010-11-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

  2. The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization.

    Science.gov (United States)

    Qi, Aisha; Yeo, Leslie; Friend, James; Ho, Jenny

    2010-02-21

    Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

  3. Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

    Science.gov (United States)

    Paulmurugan, Ramasamy; Gambhir, Sanjiv S

    2005-08-15

    Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

  4. Soluble endothelial cell-selective adhesion molecule and incident cardiovascular events in a multiethnic population.

    Science.gov (United States)

    Ren, Hao-Yu; Khera, Amit; de Lemos, James A; Ayers, Colby R; Rohatgi, Anand

    2017-09-01

    Cell adhesion molecules are key regulators of atherosclerotic plaque development, but circulating levels of soluble fragments, such as intercellular adhesion molecule (sICAM-1) and vascular cell adhesion molecule (sVCAM-1), have yielded conflicting associations with atherosclerotic cardiovascular disease (ASCVD). Endothelial cell-selective adhesion molecule (ESAM) is expressed exclusively in platelets and endothelial cells, and soluble ESAM (sESAM) levels have been associated with prevalent subclinical atherosclerosis. We therefore hypothesized that sESAM would be associated with incident ASCVD. sESAM, sICAM-1, and sVCAM-1 were measured in 2,442 participants without CVD in the Dallas Heart Study, a probability-based population sample aged 30-65 years enrolled between 2000 and 2002. ASCVD was defined as first myocardial infarction, stroke, coronary revascularization, or CV death. A total of 162 ASCVD events were analyzed over 10.4 years. Increasing sESAM was associated with ASCVD, independent of risk factors (HR Q4 vs Q1: 2.7, 95% CI 1.6-4.6). Serial adjustment for renal function, sICAM-1, VCAM-1, and prevalent coronary calcium did not attenuate these associations. Continuous ESAM demonstrated similar findings (HR 1.31, 95% CI 1.2-1.4). Addition of sESAM to traditional risk factors improved discrimination and reclassification (delta c-index: P = .009; integrated-discrimination-improvement index P = .001; net reclassification index = 0.42, 95% CI 0.15-0.68). Neither sICAM-1 nor sVCAM-1 was independently associated with ASCVD. sESAM but not sICAM-1 or sVCAM-1 levels are associated with incident ASCVD. Further studies are warranted to investigate the role of sESAM in ASCVD. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    Science.gov (United States)

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  6. CHIPMUNK: A Virtual Synthesizable Small-Molecule Library for Medicinal Chemistry, Exploitable for Protein-Protein Interaction Modulators.

    Science.gov (United States)

    Humbeck, Lina; Weigang, Sebastian; Schäfer, Till; Mutzel, Petra; Koch, Oliver

    2018-03-20

    A common issue during drug design and development is the discovery of novel scaffolds for protein targets. On the one hand the chemical space of purchasable compounds is rather limited; on the other hand artificially generated molecules suffer from a grave lack of accessibility in practice. Therefore, we generated a novel virtual library of small molecules which are synthesizable from purchasable educts, called CHIPMUNK (CHemically feasible In silico Public Molecular UNiverse Knowledge base). Altogether, CHIPMUNK covers over 95 million compounds and encompasses regions of the chemical space that are not covered by existing databases. The coverage of CHIPMUNK exceeds the chemical space spanned by the Lipinski rule of five to foster the exploration of novel and difficult target classes. The analysis of the generated property space reveals that CHIPMUNK is well suited for the design of protein-protein interaction inhibitors (PPIIs). Furthermore, a recently developed structural clustering algorithm (StruClus) for big data was used to partition the sub-libraries into meaningful subsets and assist scientists to process the large amount of data. These clustered subsets also contain the target space based on ChEMBL data which was included during clustering. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  8. Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

    Science.gov (United States)

    Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

    2014-06-01

    With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. © 2013 The Authors Biopolymers Published by Wiley Periodicals, Inc.

  9. A historical overview of protein kinases and their targeted small molecule inhibitors.

    Science.gov (United States)

    Roskoski, Robert

    2015-10-01

    Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active

  10. Evolution favors protein mutational robustness in sufficiently large populations

    Directory of Open Access Journals (Sweden)

    Venturelli Ophelia S

    2007-07-01

    Full Text Available Abstract Background An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. Results Here we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by mathematical theory, and can be quantitatively related to the way that the proteins occupy their neutral network. Conclusion Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon could contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations.

  11. Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

    Science.gov (United States)

    Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

    2017-07-21

    Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

  12. High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

    Science.gov (United States)

    Landry, James P; Fei, Yiyan; Zhu, X D

    2011-12-01

    Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

  13. Posttranslational protein S-palmitoylation and the compartmentalization of signaling molecules in neurons

    Directory of Open Access Journals (Sweden)

    SEAN I PATTERSON

    2002-01-01

    Full Text Available Protein domains play a fundamental role in the spatial and temporal organization of intracellular signaling systems. While protein phosphorylation has long been known to modify the interactions that underlie this organization, the dynamic cycling of lipids should now be included amongst the posttranslational processes determining specificity in signal transduction. The characteristics of this process are reminiscent of the properties of protein and lipid phosphorylation in determining compartmentalization through SH2 or PH domains. Recent studies have confirmed the functional importance of protein S-palmitoylation in the compartmentalization of signaling molecules that support normal physiological function in cell division and apoptosis, and synaptic transmission and neurite outgrowth. In neurons, S-palmitoylation and targeting of proteins to rafts are regulated differentially in development by a number of processes, including some related to synaptogenesis and synaptic plasticity. Alterations in the S-palmitoylation state of proteins substantially affect their cellular function, raising the possibility of new therapeutic targets in cancer and nervous system injury and disease.

  14. Chemical annotation of small and peptide-like molecules at the Protein Data Bank

    Science.gov (United States)

    Young, Jasmine Y.; Feng, Zukang; Dimitropoulos, Dimitris; Sala, Raul; Westbrook, John; Zhuravleva, Marina; Shao, Chenghua; Quesada, Martha; Peisach, Ezra; Berman, Helen M.

    2013-01-01

    Over the past decade, the number of polymers and their complexes with small molecules in the Protein Data Bank archive (PDB) has continued to increase significantly. To support scientific advancements and ensure the best quality and completeness of the data files over the next 10 years and beyond, the Worldwide PDB partnership that manages the PDB archive is developing a new deposition and annotation system. This system focuses on efficient data capture across all supported experimental methods. The new deposition and annotation system is composed of four major modules that together support all of the processing requirements for a PDB entry. In this article, we describe one such module called the Chemical Component Annotation Tool. This tool uses information from both the Chemical Component Dictionary and Biologically Interesting molecule Reference Dictionary to aid in annotation. Benchmark studies have shown that the Chemical Component Annotation Tool provides significant improvements in processing efficiency and data quality. Database URL: http://wwpdb.org PMID:24291661

  15. Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation.

    Science.gov (United States)

    Lewallen, Daniel M; Sreelatha, Anju; Dharmarajan, Venkatasubramanian; Madoux, Franck; Chase, Peter; Griffin, Patrick R; Orth, Kim; Hodder, Peter; Thompson, Paul R

    2014-02-21

    Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki's ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

  16. The chemical component dictionary: complete descriptions of constituent molecules in experimentally determined 3D macromolecules in the Protein Data Bank

    OpenAIRE

    Westbrook, John D.; Shao, Chenghua; Feng, Zukang; Zhuravleva, Marina; Velankar, Sameer; Young, Jasmine

    2014-01-01

    Summary: The Chemical Component Dictionary (CCD) is a chemical reference data resource that describes all residue and small molecule components found in Protein Data Bank (PDB) entries. The CCD contains detailed chemical descriptions for standard and modified amino acids/nucleotides, small molecule ligands and solvent molecules. Each chemical definition includes descriptions of chemical properties such as stereochemical assignments, chemical descriptors, systematic chemical names and idealize...

  17. Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

    International Nuclear Information System (INIS)

    Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

    2007-01-01

    We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

  18. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

  19. A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

    DEFF Research Database (Denmark)

    Zhang, Zhao; Hejesen, Christian; Kjelstrup, Michael Brøndum

    2014-01-01

    . The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies......Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers...

  20. Targeting the OB-Folds of Replication Protein A with Small Molecules

    Directory of Open Access Journals (Sweden)

    Victor J. Anciano Granadillo

    2010-01-01

    Full Text Available Replication protein A (RPA is the main eukaryotic single-strand (ss DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

  1. Sizing of colloidal particle and protein molecules in a hanging fluid drop

    Science.gov (United States)

    Ansari, Rafat R.; Suh, Kwang I.

    1995-01-01

    We report non-invasive particle size measurements of polystyrene latex colloidal particles and bovine serum albumin (BSA) protein molecules suspended in tiny hanging fluid drops of 30 micro-Liter volume using a newly designed fiber optic probe. The probe is based upon the principles of the technique of dynamic light scattering (DLS). The motivation for this work comes from growing protein crystals in outer space. Protein crystals have been grown previously in hanging drops in microgravity experiments on-board the space shuttle orbiter. However, obtaining quantitative information on nucleation and growth of the protein crystals in real time has always been a desired goal, but hitherto not achieved. Several protein researchers have shown interest in using DLS to monitor crystal growth process in a droplet, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. We demonstrate that such an experiment is now possible. Our system offers fast (5 seconds) determination of particle size, utilize safe levels of very low laser power (less than or equal to 0.2 mW), a small scattering volume (approximately 2 x 10(exp -5) cu mm) and high spatial coherence (Beta) values. This is a major step forward when compared to currently available DLS systems.

  2. Preference of small molecules for local minimum conformations when binding to proteins.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    2007-09-01

    Full Text Available It is well known that small molecules (ligands do not necessarily adopt their lowest potential energy conformations when binding to proteins. Analyses of protein-bound ligand crystal structures have reportedly shown that many of them do not even adopt the conformations at local minima of their potential energy surfaces (local minimum conformations. The results of these analyses raise a concern regarding the validity of virtual screening methods that use ligands in local minimum conformations. Here we report a normal-mode-analysis (NMA study of 100 crystal structures of protein-bound ligands. Our data show that the energy minimization of a ligand alone does not automatically stop at a local minimum conformation if the minimum of the potential energy surface is shallow, thus leading to the folding of the ligand. Furthermore, our data show that all 100 ligand conformations in their protein-bound ligand crystal structures are nearly identical to their local minimum conformations obtained from NMA-monitored energy minimization, suggesting that ligands prefer to adopt local minimum conformations when binding to proteins. These results both support virtual screening methods that use ligands in local minimum conformations and caution about possible adverse effect of excessive energy minimization when generating a database of ligand conformations for virtual screening.

  3. STITCH 2: an interaction network database for small molecules and proteins

    DEFF Research Database (Denmark)

    Kuhn, Michael; Szklarczyk, Damian; Franceschini, Andrea

    2010-01-01

    Over the last years, the publicly available knowledge on interactions between small molecules and proteins has been steadily increasing. To create a network of interactions, STITCH aims to integrate the data dispersed over the literature and various databases of biological pathways, drug......-target relationships and binding affinities. In STITCH 2, the number of relevant interactions is increased by incorporation of BindingDB, PharmGKB and the Comparative Toxicogenomics Database. The resulting network can be explored interactively or used as the basis for large-scale analyses. To facilitate links to other...... chemical databases, we adopt InChIKeys that allow identification of chemicals with a short, checksum-like string. STITCH 2.0 connects proteins from 630 organisms to over 74,000 different chemicals, including 2200 drugs. STITCH can be accessed at http://stitch.embl.de/....

  4. Measuring the force of single protein molecule detachment from surfaces with AFM.

    Science.gov (United States)

    Tsapikouni, Theodora S; Missirlis, Yannis F

    2010-01-01

    Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

  5. Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

    Science.gov (United States)

    Hsueh, Carlin

    Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

  6. Pulsed laser study of excited states of aromatic molecules absorbed in globular proteins

    International Nuclear Information System (INIS)

    Cooper, M.; Thomas, J.K.

    1977-01-01

    Pyrene and several derivatives of pyrene such as pyrene sulfonic acid, and pyrene butyric acid were incorporated into bovine serum albumin (BSA) in aqueous solution. The pyrene chromophore was subsequently excited by a pulse of uv light (lambda = 3471 A) from a Q switched frequency doubled ruby laser. The lifetime of the pyrene excited singlet and triplet states were monitored by time resolved spectrophotometry. Various molecules, such as O 2 and I - , dissolved in the aqueous phase, diffused into the protein and quenched pyrene excited states. The rates of these reactions were followed under a variety of conditions such as pH and temperature and in the presence of inert additives. The rates of pyrene excited-state quenching were often considerably smaller than the rates observed in simple solutions. A comparison of the rates in the protein and homogeneous solutions gives information on the factors such as temperature, charge, and pH that control the movement of small molecules in and into BSA

  7. How Diverse are the Protein-Bound Conformations of Small-Molecule Drugs and Cofactors?

    Science.gov (United States)

    Friedrich, Nils-Ole; Simsir, Méliné; Kirchmair, Johannes

    2018-03-01

    Knowledge of the bioactive conformations of small molecules or the ability to predict them with theoretical methods is of key importance to the design of bioactive compounds such as drugs, agrochemicals and cosmetics. Using an elaborate cheminformatics pipeline, which also evaluates the support of individual atom coordinates by the measured electron density, we compiled a complete set (“Sperrylite Dataset”) of high-quality structures of protein-bound ligand conformations from the PDB. The Sperrylite Dataset consists of a total of 10,936 high-quality structures of 4548 unique ligands. Based on this dataset, we assessed the variability of the bioactive conformations of 91 small molecules—each represented by a minimum of ten structures—and found it to be largely independent of the number of rotatable bonds. Sixty-nine molecules had at least two distinct conformations (defined by an RMSD greater than 1 Å). For a representative subset of 17 approved drugs and cofactors we observed a clear trend for the formation of few clusters of highly similar conformers. Even for proteins that share a very low sequence identity, ligands were regularly found to adopt similar conformations. For cofactors, a clear trend for extended conformations was measured, although in few cases also coiled conformers were observed. The Sperrylite Dataset is available for download from http://www.zbh.uni-hamburg.de/sperrylite_dataset.

  8. Solvent friction effects propagate over the entire protein molecule through low-frequency collective modes.

    Science.gov (United States)

    Moritsugu, Kei; Kidera, Akinori; Smith, Jeremy C

    2014-07-24

    Protein solvation dynamics has been investigated using atom-dependent Langevin friction coefficients derived directly from molecular dynamics (MD) simulations. To determine the effect of solvation on the atomic friction coefficients, solution and vacuum MD simulations were performed for lysozyme and staphylococcal nuclease and analyzed by Langevin mode analysis. The coefficients thus derived are roughly correlated with the atomic solvent-accessible surface area (ASA), as expected from the fact that friction occurs as the result of collisions with solvent molecules. However, a considerable number of atoms with higher friction coefficients are found inside the core region. Hence, the influence of solvent friction propagates into the protein core. The internal coefficients have large contributions from the low-frequency modes, yielding a simple picture of the surface-to-core long-range damping via solvation governed by collective low-frequency modes. To make use of these findings in implicit-solvent modeling, we compare the all-atom friction results with those obtained using Langevin dynamics (LD) with two empirical representations: the constant-friction and the ASA-dependent (Pastor-Karplus) friction models. The constant-friction model overestimates the core and underestimates the surface damping whereas the ASA-dependent friction model, which damps protein atoms only on the solvent-accessible surface, reproduces well the friction coefficients for both the surface and core regions observed in the explicit-solvent MD simulations. Therefore, in LD simulation, the solvent friction coefficients should be imposed only on the protein surface.

  9. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

    Science.gov (United States)

    Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

    2003-10-01

    Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

  10. Hyccin, the Molecule Mutated in the Leukodystrophy Hypomyelination and Congenital Cataract (HCC), Is a Neuronal Protein

    Science.gov (United States)

    Giacomini, Caterina; Musante, Veronica; Fruscione, Floriana; La Padula, Veronica; Biancheri, Roberta; Scarfì, Sonia; Prada, Valeria; Sotgia, Federica; Duncan, Ian D.; Zara, Federico; Werner, Hauke B.; Lisanti, Michael P.; Nobbio, Lucilla; Corradi, Anna; Minetti, Carlo

    2012-01-01

    “Hypomyelination and Congenital Cataract”, HCC (MIM #610532), is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS). Using heterozygous mice expressing the b-galactosidase (LacZ) gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10) and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC)/Dorsal Root Ganglion (DRG) sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination. PMID:22461884

  11. Hyccin, the molecule mutated in the leukodystrophy hypomyelination and congenital cataract (HCC, is a neuronal protein.

    Directory of Open Access Journals (Sweden)

    Elisabetta Gazzerro

    Full Text Available "Hypomyelination and Congenital Cataract", HCC (MIM #610532, is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS. Using heterozygous mice expressing the b-galactosidase (LacZ gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10 and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC/Dorsal Root Ganglion (DRG sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination.

  12. Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells.

    Science.gov (United States)

    Jordheim, Lars Petter; Barakat, Khaled H; Heinrich-Balard, Laurence; Matera, Eva-Laure; Cros-Perrial, Emeline; Bouledrak, Karima; El Sabeh, Rana; Perez-Pineiro, Rolando; Wishart, David S; Cohen, Richard; Tuszynski, Jack; Dumontet, Charles

    2013-07-01

    The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl

  13. The Relationship Between Low-Frequency Motions and Community Structure of Residue Network in Protein Molecules.

    Science.gov (United States)

    Sun, Weitao

    2018-01-01

    The global shape of a protein molecule is believed to be dominant in determining low-frequency deformational motions. However, how structure dynamics relies on residue interactions remains largely unknown. The global residue community structure and the local residue interactions are two important coexisting factors imposing significant effects on low-frequency normal modes. In this work, an algorithm for community structure partition is proposed by integrating Miyazawa-Jernigan empirical potential energy as edge weight. A sensitivity parameter is defined to measure the effect of local residue interaction on low-frequency movement. We show that community structure is a more fundamental feature of residue contact networks. Moreover, we surprisingly find that low-frequency normal mode eigenvectors are sensitive to some local critical residue interaction pairs (CRIPs). A fair amount of CRIPs act as bridges and hold distributed structure components into a unified tertiary structure by bonding nearby communities. Community structure analysis and CRIP detection of 116 catalytic proteins reveal that breaking up of a CRIP can cause low-frequency allosteric movement of a residue at the far side of protein structure. The results imply that community structure and CRIP may be the structural basis for low-frequency motions.

  14. High-throughput single-molecule force spectroscopy for membrane proteins

    Science.gov (United States)

    Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

    2008-09-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  15. High-throughput single-molecule force spectroscopy for membrane proteins

    International Nuclear Information System (INIS)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios; Ratera, Merce; Palacin, Manuel; Bippes, Christian A; Mueller, Daniel J

    2008-01-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications

  16. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  17. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  18. Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray

    Energy Technology Data Exchange (ETDEWEB)

    Ichiyanagi, Kouhei; Sasaki, Yuji C. [Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 609 Kiban Building 5-1-5 Kashiwanoha, Kahiwashi, Chiba 277-8561 (Japan); Japan Science and Technology Agency, CREST, CREST, Sasaki-Team, 609 Kiban Building, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Sekiguchi, Hiroshi; Hoshino, Masato; Kajiwara, Kentaro; Senba, Yasunori; Ohashi, Haruhiko; Ohta, Noboru [Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan); Hoshisashi, Kentaro; Jae-won, Chang; Tokue, Maki; Matsushita, Yufuku [Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 609 Kiban Building 5-1-5 Kashiwanoha, Kahiwashi, Chiba 277-8561 (Japan); Nishijima, Masaki; Inoue, Yoshihisa [Department of Applied Chemistry and Office for University-Industry Collaboration, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yagi, Naoto [Japan Science and Technology Agency, CREST, CREST, Sasaki-Team, 609 Kiban Building, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2013-10-15

    Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10–20 keV (△E/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

  19. Immunolocalization of notch signaling protein molecules in a maxillary chondrosarcoma and its recurrent tumor

    Directory of Open Access Journals (Sweden)

    Siar CH

    2010-10-01

    Full Text Available Abstract Background Notch receptors are critical determinants of cell fate in a variety of organisms. Notch signaling is involved in the chondrogenic specification of neural crest cells. Aberrant Notch activity has been implicated in numerous human diseases including cancers; however its role in chondrogenic tumors has not been clarified. Method Tissue samples from a case of primary chondrosarcoma of the maxilla and its recurrent tumor were examined immunohistochemically for Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1 expression. Results Both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I. Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of Jagged1, and weak to absent staining for Notch1, 4 and Delta1. Protein immunolocalization was largely membranous and cytoplasmic, sometimes outlining the lacunae of malignant chondrocytes. Hyaline cartilage demonstrated a diffuse or granular precipitation of Jagged1 suggesting presence of soluble Jagged1 activity at sites of abnormal chondrogenesis. No immunoreactivity for the other Notch members was observed. Calcified cartilage was consistently Notch-negative indicating down-regulation of Notch with cartilage maturation. Stromal components namely endothelial cells and fibroblasts variably expressed Notch1, 3 and Jagged1 but were mildly or non-reactive for the other members. Conclusions Results indicate that Notch signaling pathway may participate in cellular differentiation and proliferation in chondrosarcoma. Findings implicate Notch3 and Jagged1 as key molecules that influence the differentiation and maturation of cells of chondrogenic lineage.

  20. Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions.

    Science.gov (United States)

    Götz, Markus; Wortmann, Philipp; Schmid, Sonja; Hugel, Thorsten

    2018-01-30

    Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

  1. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p: reinterpretation of recent single molecule experiments.

    Science.gov (United States)

    Stigter, Dirk

    2004-07-01

    Brewer et al. (Biophys. J. 85 (2003) 2519-2524) have studied the compaction of dsDNA in a double flow cell by observing the extension of stained DNA tethered in buffer solutions with or without Abf2p. They use a Langmuir adsorption model in which one Abf2p molecule adsorbs on one site on the DNA, and the binding constant, K, is given as the ratio of the experimental rates of adsorption and desorption. This paper presents an improved interpretation. Instead of Langmuir adsorption we use the more appropriate McGhee-von Hippel (J. Mol. Biol. 86 (1974) 469-489) theory for the adsorption of large ligands to a one-dimensional lattice. We assume that each adsorbed molecule shortens the effective contour length of DNA by the foot print of Abf2p of 27 base pairs. When Abf2p adsorbs to DNA stretched in the flowing buffer solution, we account for a tension effect that decreases the adsorption rate and the binding constant by a factor of 2 to 4. The data suggest that the accessibility to Abf2p decreases significantly with increasing compaction of DNA, resulting in a lower adsorption rate and a lower binding constant. The kinetics reported by Brewer et al. (Biophys. J. 85 (2003) 2519-2524) lead to a binding constant K=3.6 x 10(6) M(-1) at the beginning, and to K=5 x 10(5) M(-1) near the end of a compaction run, more than an order of magnitude lower than the value K=2.57 x 10(7) M(-1) calculated by Brewer et al. (Biophys. J. 85 (2003) 2519-2524).

  2. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    International Nuclear Information System (INIS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-01-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions

  3. Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

    Science.gov (United States)

    Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A

    2011-04-28

    Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded

  4. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Perry

    Full Text Available Ubiquitin (Ub is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs. However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1, a critical mediator of the unfolded protein response (UPR. WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1 through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

  5. Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

    Science.gov (United States)

    Hokari, Ryota; Kitagawa, Noritake; Watanabe, Chikako; Komoto, Shunsuke; Kurihara, Chie; Okada, Yoshikiyo; Kawaguchi, Atsushi; Nagao, Shigeaki; Hibi, Toshifumi; Miura, Soichiro

    2008-07-01

    Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

  6. The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

    Science.gov (United States)

    Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

    2016-02-01

    Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

  7. Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

    Science.gov (United States)

    Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

    2010-01-01

    We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

  8. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

    Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  9. Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

    Science.gov (United States)

    Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

    2017-04-28

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

  10. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins

    DEFF Research Database (Denmark)

    Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin are all complement activating soluble pattern recognition molecules with recognition domains linked to collagen-like regions. All four may form complexes with four structurally related proteins, the three MBL-associated serine...... proteases (MASPs), MASP-1, MASP-2 and MASP-3, and a smaller MBL-associated protein (MAp19). The four recognition molecules recognize patterns of carbohydrate or acetyl-group containing ligands. After binding to the relevant targets all four are able to activate the complement system. We thus have a system...... where four different and/or overlapping patterns of microbial origin or patterns of altered-self may be recognized, but in all cases the signalling molecules, the MASPs, are shared. MASP-1 and MASP-3 are formed from one gene, MASP1/3, by alternative splicing generating two different mRNAs from a single...

  11. The mechanism and properties of bio-photon emission and absorption in protein molecules in living systems

    Science.gov (United States)

    Pang, Xiao-feng

    2012-05-01

    The mechanism and properties of bio-photon emission and absorption in bio-tissues were studied using Pang's theory of bio-energy transport, in which the energy spectra of protein molecules are obtained from the discrete dynamic equation. From the energy spectra, it was determined that the protein molecules could both radiate and absorb bio-photons with wavelengths of energy level transitions of the excitons. These results were consistent with the experimental data; this consisted of infrared absorption data from collagen, bovine serum albumin, the protein-like molecule acetanilide, plasma, and a person's finger, and the laser-Raman spectra of acidity I-type collagen in the lungs of a mouse, and metabolically active Escherichia coli. We further elucidated the mechanism responsible for the non-thermal biological effects produced by the infrared light absorbed by the bio-tissues, using the above results. No temperature rise was observed; instead, the absorbed infrared light promoted the vibrations of amides as well the transport of the bio-energy from one place to other in the protein molecules, which changed their conformations. These experimental results, therefore, not only confirmed the validity of the mechanism of bio-photon emission, and the newly developed theory of bio-energy transport mentioned above, but also explained the mechanism and properties of the non-thermal biological effects produced by the absorption of infrared light by the living systems.

  12. Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

    Science.gov (United States)

    Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

    2014-01-01

    Homologous recombination is a conserved pathway for repairing double–stranded breaks, which are processed to yield single–stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single–molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Together, our work illustrates the spatial and temporal progression of RPA and Rad52 association with the presynaptic complex, and reveals a novel RPA–Rad52–Rad51–ssDNA intermediate, which has implications for understanding how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages recombination. PMID:25195049

  13. The consequences of translational and rotational entropy lost by small molecules on binding to proteins

    Science.gov (United States)

    Murray, Christopher W.; Verdonk, Marcel L.

    2002-10-01

    When a small molecule binds to a protein, it loses a significant amount of rigid body translational and rotational entropy. Estimates of the associated energy barrier vary widely in the literature yet accurate estimates are important in the interpretation of results from fragment-based drug discovery techniques. This paper describes an analysis that allows the estimation of the rigid body entropy barrier from the increase in binding affinities that results when two fragments of known affinity and known binding mode are joined together. The paper reviews the relatively rare number of examples where good quality data is available. From the analysis of this data, we estimate that the barrier to binding, due to the loss of rigid-body entropy, is 15-20 kJ/mol, i.e. around 3 orders of magnitude in affinity at 298 K. This large barrier explains why it is comparatively rare to observe multiple fragments binding to non-overlapping adjacent sites in enzymes. The barrier is also consistent with medicinal chemistry experience where small changes in the critical binding regions of ligands are often poorly tolerated by enzymes.

  14. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

    Science.gov (United States)

    Bell, Nicholas A. W.; Keyser, Ulrich F.

    2016-07-01

    The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

  15. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  16. Protein electrochemistry using graphene-based nano-assembly: an ultrasensitive electrochemical detection of protein molecules via nanoparticle-electrode collisions.

    Science.gov (United States)

    Li, Da; Liu, Jingquan; Barrow, Colin J; Yang, Wenrong

    2014-08-04

    We describe a new electrochemical detection approach towards single protein molecules (microperoxidase-11, MP-11), which are attached to the surface of graphene nanosheets. The non-covalently functionalized graphene nanosheets exhibit enhanced electroactive surface area, where amplified redox current is produced when graphene nanosheets collide with the electrode.

  17. Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.

    Science.gov (United States)

    Kanoh, Naoki; Asami, Aya; Kawatani, Makoto; Honda, Kaori; Kumashiro, Saori; Takayama, Hiroshi; Simizu, Siro; Amemiya, Tomoyuki; Kondoh, Yasumitsu; Hatakeyama, Satoru; Tsuganezawa, Keiko; Utata, Rei; Tanaka, Akiko; Yokoyama, Shigeyuki; Tashiro, Hideo; Osada, Hiroyuki

    2006-12-18

    We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.

  18. System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

    Science.gov (United States)

    Malina, Halina Z

    2011-01-19

    The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules

  19. Peculiarities of the introduction of technetium isotopes into protein molecules - of human serum albumin as an example

    International Nuclear Information System (INIS)

    Stanko, V.I.; Ovsyannikov, N.N.; Zuykova, N.P.; Gouskov, A.F.; Kovalchouk, N.D.

    1978-07-01

    Pecularities of the introduction of the radioisotope technetium 99m into the molecules of human serum albumin have been investigated. Tin not only participates in the Tc(V11) reduction process, but is incorporated into the originating Tc albumin complex. It is shown that no more than four technetium atoms enter into bond with an albumin molecule. The authors express their opinion that in order to produce high-quality protein preparations, the albumin has to be modified through a polyfunctional complexing agent which forms an entirely saturated coordination complex with Tc(IV)

  20. Pecularities of the introduction of technetium isotopes into protein molecules using human serum albumin as an example

    International Nuclear Information System (INIS)

    Stanko, V.I.; Ovsyannikov, N.N.; Sujkova, N.P.; Gus'kov, A.F.; Koval'chuk, N.D.

    1978-01-01

    Pecularities of the introduction of the radioisotope technetium 99m into the molecules of human serum albumin have been investigated. Tin not only participates in the Tc(VII) reduction process, but is incorporated into the originating Tc albumin complex. It is shown that no more than four technetium atoms enter into bond with an albumin molecule. The authors express their opinion that in order to produce high-quality protein preparations, the albumin has to be modified through a polyfunctional complexing agent which forms an entirely saturated coordination complex with Tc(IV). (author)

  1. Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

    interactions, thereby modulating a range of biological processes. This review summarizes interactions between NCAM and other CAMs and ECM proteins. Additionally, the role of NCAM as a receptor for rabies virus, and its implications in rabies infections is briefly described. Interactions between NCAM and its...

  2. A novel small molecule inhibitor of the DNA repair protein Ku70/80.

    Science.gov (United States)

    Weterings, Eric; Gallegos, Alfred C; Dominick, Lauren N; Cooke, Laurence S; Bartels, Trace N; Vagner, Josef; Matsunaga, Terry O; Mahadevan, Daruka

    2016-07-01

    Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents. Copyright © 2016 Elsevier B.V. All

  3. Thomas-Reiche-Khun populations in X-CH 3 and X-C 2H 5 series of molecules

    Science.gov (United States)

    Zitto, M. E.; Caputo, M. C.; Ferraro, M. B.; Lazzeretti, P.

    2000-09-01

    Calculations of nuclear electric shieldings, equivalent to dipole moment geometric derivatives, and related to atomic polar tensors, are presented for X-CH 3 and X-C 2H 5 molecules with X=NH 2, OH and F. The electric shielding tensors satisfy a constraint for the electrostatic equilibrium, i.e., the mixed length-acceleration Thomas-Reiche-Khun sum rule, which gives important indications on the reliability of theoretical predictions of IR intensities and leads to the definition of atomic populations. Numerical evidence was found for the additivity and transferability of atomic populations, within the X-substituted alkane series.

  4. Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

    Science.gov (United States)

    Wollman, Adam J M; Leake, Mark C

    2015-01-01

    We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of

  5. Monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in exudative age-related macular degeneration.

    Science.gov (United States)

    Jonas, Jost B; Tao, Yong; Neumaier, Michael; Findeisen, Peter

    2010-10-01

    To examine intraocular concentrations of monocyte chemoattractant protein 1 (MCP-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and vascular endothelial growth factor (VEGF) in eyes with exudative age-related macular degeneration (AMD). The investigation included a study group of 28 patients (28 eyes) with exudative AMD and a control group of 25 patients (25 eyes) with cataract. The concentrations of MCP-1, sICAM-1, sVCAM-1, and VEGF in aqueous humor samples obtained during surgery were measured using a solid-phase chemiluminescence immunoassay. The study group as compared with the control group had higher aqueous concentrations of sICAM-1 (mean [SD], 844 [2073] vs 246 [206] pg/mL, respectively; P < .001), sVCAM-1 (mean [SD], 7978 [7120] vs 2999 [1426] pg/mL, respectively; P < .001), and MCP-1 (mean [SD], 587 [338] vs 435 [221] pg/mL, respectively; P = .07). The concentration of VEGF did not vary significantly between the groups (P = .76). The MCP-1 concentration was significantly associated with macular thickness (r = 0.40; P = .004). It decreased significantly with the type of subfoveal neovascular membrane (classic membrane type, occult membrane, retinal pigment epithelium detachment) (P = .009). The concentrations of sICAM-1, sVCAM-1, and VEGF were not significantly associated with membrane type and macular thickness (P ≥ .18). Concentrations of MCP-1, sICAM-1, and sVCAM-1 are significantly associated with exudative AMD, even in the presence of normal VEGF concentrations. Intraocular MCP-1 concentrations are correlated with the subfoveal neovascular membrane type and the amount of macular edema. One may infer that MCP-1, sICAM-1, and sVCAM-1 could potentially be additional target molecules in therapy for exudative AMD.

  6. Characterization of Small Molecule Scaffolds that Bind to the Shigella Type III Secretion System Protein IpaD

    Science.gov (United States)

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E.; De Guzman, Roberto N.

    2017-01-01

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salts sterols. Here, we identified four new small molecule scaffolds that bind to IpaD based on the methylquinoline, pyrrolidin-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small molecule inhibitors of IpaD that could lead to new anti-infectives. PMID:28750143

  7. Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD.

    Science.gov (United States)

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E; De Guzman, Roberto N

    2017-09-21

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. An ultra-HTS process for the identification of small molecule modulators of orphan G-protein-coupled receptors.

    Science.gov (United States)

    Cacace, Angela; Banks, Martyn; Spicer, Timothy; Civoli, Francesca; Watson, John

    2003-09-01

    G-protein-coupled receptors (GPCRs) are the most successful target proteins for drug discovery research to date. More than 150 orphan GPCRs of potential therapeutic interest have been identified for which no activating ligands or biological functions are known. One of the greatest challenges in the pharmaceutical industry is to link these orphan GPCRs with human diseases. Highly automated parallel approaches that integrate ultra-high throughput and focused screening can be used to identify small molecule modulators of orphan GPCRs. These small molecules can then be employed as pharmacological tools to explore the function of orphan receptors in models of human disease. In this review, we describe methods that utilize powerful ultra-high-throughput screening technologies to identify surrogate ligands of orphan GPCRs.

  9. Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor

    Directory of Open Access Journals (Sweden)

    Sandra Posch

    2016-09-01

    Full Text Available We here give information for a deeper understanding of single molecule force spectroscopy (SMFS data through the example of the blood protein von Willebrand factor (VWF. It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK was used for this demonstration. Further, Brownian dynamics (BD simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, “Mutual A domain interactions in the force sensing protein von Willebrand Factor” (Posch et al., 2016 [1]. Keywords: Atomic force microscopy, Single molecule force spectroscopy, Molecular dynamics simulation, Brownian dynamics simulation, von Willebrand factor

  10. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, Sourav, E-mail: sourav.kunduphy@gmail.com; Karmakar, S.N.

    2016-07-15

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  11. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

    Science.gov (United States)

    Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

    2014-03-21

    A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

  12. Steering wave packet dynamics and population transfer between electronic states of the Na2 molecule by femtosecond laser pulses

    International Nuclear Information System (INIS)

    Yuan Kaijun; Sun Zhigang; Cong Shulin; Wang Senming; Yu Jie; Lou Nanquan

    2005-01-01

    An approach used for steering the wave packet dynamics and the population transfer between electronic states of the Na 2 molecule by a pair of femtosecond laser pulses is demonstrated. Four controlling schemes, i.e., four different combinations of time delays (intuitive and counterintuitive sequences) and frequency detunings (positive and negative detunings), are discussed in detail. The light-induced potentials are used to describe the wave packet dynamics and population transfer. The numerical results show that the wave packet excited by femtosecond laser pulses oscillates drastically on 2 1 Π g state with time. The efficiency of controlling population transfer from the X 1 Σ g + to2 1 Π g states of Na 2 is nearly 100% for the schemes of the counterintuitive sequence pulses with positive and negative detunings

  13. Fast electron transfer through a single molecule natively structured redox protein

    DEFF Research Database (Denmark)

    Della Pia, Eduardo Antonio; Chi, Qijin; Macdonald, J. Emyr

    2012-01-01

    The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conduc...

  14. Molecules and morphology reveal overlooked populations of two presumed extinct Australian sea snakes (Aipysurus: Hydrophiinae)

    DEFF Research Database (Denmark)

    Sanders, Kate Laura; Schroeder, Tina; Guinea, Michael L.

    2015-01-01

    in northwest Australia are widely threatened by infrastructure developments and sea snakes are presently omitted from environmental impact assessments for industry. Further studies are urgently needed to assess these species’ remaining distributions, population structure, and extent of occurrence in protected...

  15. The effect of soy protein beverages on serum cell adhesion molecule concentrations in prehypertensive/stage 1 hypertensive individuals.

    Science.gov (United States)

    Dettmer, Michelle; Alekel, D Lee; Lasrado, Joanne A; Messina, Mark; Carriquiry, Alicia; Heiberger, Kevin; Stewart, Jeanne W; Franke, Warren

    2012-04-01

    Prehypertensive and hypertensive individuals are at increased risk of atherosclerotic cardiovascular disease (CVD), in part because hypertension contributes to endothelial dysfunction and increased cell adhesion molecule expression. Soy protein and isoflavones may favorably alter CVD risk factors, and hence the aim of this study was to determine whether intake of cow's milk compared with soy beverage prepared from whole soy bean (WSB) or soy protein isolate (SPI) would lower soluble cell adhesion molecule concentrations as a means of decreasing CVD risk. We enrolled healthy prehypertensive/stage 1 hypertensive men (n = 60; 18-63 years) and premenopausal women (n = 8; 20-48 years). Participants were randomized to 1 of 3 groups for 8 weeks: cow's milk (600 mL/d), SPI beverage (840 mL/d; 30.1 mg total isoflavones/d), or WSB beverage (840 mL/d; 91.4 mg total isoflavones/d). We measured soluble vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin) concentrations at baseline and week 8. Soluble CAM concentrations were not altered by treatment and did not differ between prehypertensive and hypertensive participants. However, analysis of variance indicated a treatment × gender interaction (gender effect) for ICAM-1 (p = 0.0037) but not for E-selectin (p = 0.067) or VCAM-1 (p = 0.16). Men had higher concentrations of ICAM-1 and E-selectin, respectively, at baseline (p = 0.0071, p = 0.049) and week 8 (p = 0.0054, p = 0.038) than women did. Neither intake of cow's milk nor soy beverage for 8 weeks altered soluble CAM concentrations in prehypertensive/stage 1 hypertensive individuals, suggesting that neither type of beverage diminished atherosclerotic CVD risk in mildly hypertensive individuals by way of improving circulating CAM concentrations.

  16. Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

    Science.gov (United States)

    Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

    2014-07-29

    The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules

    NARCIS (Netherlands)

    Martin, A.H.; Cohen Stuart, M.A.; Bos, M.A.; Vliet, T. van

    2005-01-01

    The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, σf,

  18. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

    Science.gov (United States)

    Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin

    2012-01-01

    Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740

  19. The hyaluronan and proteoglycan link proteins: Organizers of the brain extracellular matrix and key molecules for neuronal function and plasticity.

    Science.gov (United States)

    Oohashi, Toshitaka; Edamatsu, Midori; Bekku, Yoko; Carulli, Daniela

    2015-12-01

    The hyaluronan and proteoglycanbinding link protein (Hapln) is a key molecule in the formation and control of hyaluronan-based condensed perineuronal matrix in the adult brain. This review summarizes the recent advances in understanding the role of Haplns in the formation and control of two distinct types of perineuronal matrices, one for "classical" PNN and the other for the specialized extracellular matrix (ECM) at the node of Ranvier in the central nervous system (CNS). We introduce the structural components of each ECM organization including the basic concept of supramolecular structure named "HLT model". We furthermore summarize the developmental and physiological role of perineuronal ECMs from the studies of Haplns and related molecules. Finally, we also discuss the potential mechanism modulating PNNs in the adult CNS. This layer of organized matrices may exert a direct effect via core protein or sugar moiety from the structure or by acting as a binding site for biologically active molecules, which are important for neuronal plasticity and saltatory conduction. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. A Fragment-Based Method of Creating Small-Molecule Libraries to Target the Aggregation of Intrinsically Disordered Proteins.

    Science.gov (United States)

    Joshi, Priyanka; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P J; Dobson, Christopher M; Vendruscolo, Michele

    2016-03-14

    The aggregation process of intrinsically disordered proteins (IDPs) has been associated with a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Currently, however, no drug in clinical use targets IDP aggregation. To facilitate drug discovery programs in this important and challenging area, we describe a fragment-based approach of generating small-molecule libraries that target specific IDPs. The method is based on the use of molecular fragments extracted from compounds reported in the literature to inhibit of the aggregation of IDPs. These fragments are used to screen existing large generic libraries of small molecules to form smaller libraries specific for given IDPs. We illustrate this approach by describing three distinct small-molecule libraries to target, Aβ, tau, and α-synuclein, which are three IDPs implicated in Alzheimer's and Parkinson's diseases. The strategy described here offers novel opportunities for the identification of effective molecular scaffolds for drug discovery for neurodegenerative disorders and to provide insights into the mechanism of small-molecule binding to IDPs.

  1. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    International Nuclear Information System (INIS)

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu

    2007-01-01

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-κB activation and nuclear translocation in an IκBα-dependent manner. The inhibitory effects were associated with reduction of inhibitor IκB kinase activity and stabilization of the NF-κB inhibitor IκB. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations

  2. New Ionization and Detection Technologies for Mass Spectrometry Imaging. From Small Molecules to Intact Proteins

    NARCIS (Netherlands)

    Kiss, A.

    2014-01-01

    There is a constantly growing interest in biomedical research to visualize changes in the location of various biomolecules in tissue sections as a result of complex diseases. Mass spectrometry imaging is one of the techniques that enable the mapping of molecules on a 2D surface. However, the

  3. Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration

    Directory of Open Access Journals (Sweden)

    A.T. Pettersson-Klein

    2018-03-01

    Full Text Available Objective: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1 regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. Methods: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. Results: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. Conclusions: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders. Keywords: Small molecule screening, PGC-1a, PGC-1alpha, PGC-1alpha1, Protein stabilization, UCP1, Mitochondrial respiration, Brown adipose tissue

  4. Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization.

    Science.gov (United States)

    Beuming, Thijs; Che, Ye; Abel, Robert; Kim, Byungchan; Shanmugasundaram, Veerabahu; Sherman, Woody

    2012-03-01

    Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts. Copyright © 2011 Wiley Periodicals, Inc.

  5. Calculation of Ground State Rotational Populations for Kinetic Gas Homonuclear Diatomic Molecules including Electron-Impact Excitation and Wall Collisions

    International Nuclear Information System (INIS)

    Farley, David R.

    2010-01-01

    A model has been developed to calculate the ground-state rotational populations of homonuclear diatomic molecules in kinetic gases, including the effects of electron-impact excitation, wall collisions, and gas feed rate. The equations are exact within the accuracy of the cross sections used and of the assumed equilibrating effect of wall collisions. It is found that the inflow of feed gas and equilibrating wall collisions can significantly affect the rotational distribution in competition with non-equilibrating electron-impact effects. The resulting steady-state rotational distributions are generally Boltzmann for N (ge) 3, with a rotational temperature between the wall and feed gas temperatures. The N = 0,1,2 rotational level populations depend sensitively on the relative rates of electron-impact excitation versus wall collision and gas feed rates.

  6. Lack of Population Structure in Coriander Populations Based on SDS (Seed Storage Protein Page Analysis

    Directory of Open Access Journals (Sweden)

    Gülsüm Yaldiz

    2016-08-01

    Full Text Available Genetic variation is prerequisite for plant breeding. Nothing information existed in the literature for available diversity of Coriander accession in Turkey. Plant breeding activities are negligible in Turkey. So in order to start effective plant breeding program in Turkey, information on the available genetic diversity is viable. Therefore we planned to study the genetic variation and population structure of 29 Coriander accessions by seed storage protein (SDS. SDS analysis elaborated the lack of population structure and genetic bottleneck in the Coriander accessions in Turkey. Based on the results of this study, it was clear that sampling strategy was not appropriate and plant introduction should be made from different sources and diverse genotypes should be used as parents to initialize the effective Turkish Coriander breeding program.

  7. A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules.

    Science.gov (United States)

    Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

    2014-11-15

    The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules

    NARCIS (Netherlands)

    Schnettler, E.; Hemmes, J.C.; Huisman, R.; Goldbach, R.W.; Prins, M.W.; Kormelink, R.J.M.

    2010-01-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs

  9. Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin.

    Science.gov (United States)

    Udani, M; Zen, Q; Cottman, M; Leonard, N; Jefferson, S; Daymont, C; Truskey, G; Telen, M J

    1998-01-01

    Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors. PMID:9616226

  10. Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

    Science.gov (United States)

    Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

    2008-12-01

    Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

  11. Biotin conjugated organic molecules and proteins for cancer therapy: A review.

    Science.gov (United States)

    Maiti, Santanu; Paira, Priyankar

    2018-02-10

    The main transporter for biotin is sodium dependent multivitamin transporter (SMVT), which is overexpressed in various aggressive cancer cell lines such as ovarian (OV 2008, ID8), leukemia (L1210FR), mastocytoma (P815), colon (Colo-26), breast (4T1, JC, MMT06056), renal (RENCA, RD0995), and lung (M109) cancer cell lines. Furthermore, its overexpression was found higher to that of folate receptor. Therefore, biotin demand in the rapidly growing tumors is higher than normal tissues. Several biotin conjugated organic molecules has been reported here for selective delivery of the drug in cancer cell. Biotin conjugated molecules are showing higher fold of cytotoxicity in biotin positive cancer cell lines than the normal cell. Nanoparticles and polymer surface modified drugs and biotin mediated cancer theranostic strategy was highlighted in this review. The cytotoxicity and selectivity of the drug in cancer cells has enhanced after biotin conjugation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  12. Single-Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2.

    Science.gov (United States)

    Lei, Hai; He, Chengzhi; Hu, Chunguang; Li, Jinliang; Hu, Xiaodong; Hu, Xiaotang; Li, Hongbin

    2017-05-22

    Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2) 4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2) 4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes.

    Science.gov (United States)

    Kerekov, Nikola S; Ivanova, Iva I; Mihaylova, Nikolina M; Nikolova, Maria; Prechl, Jozsef; Tchorbanov, Andrey I

    2014-10-01

    Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

  14. A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kimori, Yoshitaka [Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502 (Japan); Oguchi, Yosuke [Department of Electric Engineering, Kogakuin University, Hachioji, Tokyo 192-0015 (Japan); Ichise, Norihiko [Department of Visual Communication, Komazawa Women' s University, Inagi, Tokyo 206-8511 (Japan); Baba, Norio [Department of Electric Engineering, Kogakuin University, Hachioji, Tokyo 192-0015 (Japan); Katayama, Eisaku [Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)]. E-mail: ekatayam@ims.u-tokyo.ac.jp

    2007-01-15

    Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the 'Structural analysis of single molecules', we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application.

  15. A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy

    International Nuclear Information System (INIS)

    Kimori, Yoshitaka; Oguchi, Yosuke; Ichise, Norihiko; Baba, Norio; Katayama, Eisaku

    2007-01-01

    Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the 'Structural analysis of single molecules', we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application

  16. Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

    Science.gov (United States)

    Maity, Hiranmay; Reddy, Govardhan

    2018-04-01

    Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

  17. Promiscuity and the conformational rearrangement of drug-like molecules: insight from the protein data bank.

    Science.gov (United States)

    He, Michael W; Lee, Patrick S; Sweeney, Zachary K

    2015-02-01

    Selectivity is a central aspect of lead optimization in the drug discovery process. Medicinal chemists often try to decrease molecular flexibility to improve selectivity, given the common belief that the two are interdependent. To investigate the relationship between polypharmacology and conformational flexibility, we mined the Protein Data Bank and constructed a dataset of pharmaceutically relevant ligands that crystallized in more than one protein target while binding to each co-crystallized receptor with similar in vitro affinities. After analyzing the molecular conformations of these 100 ligands, we found that 59 ligands bound to different protein targets without significantly changing conformation, suggesting that there is no distinct correlation between conformational flexibility and polypharmacology within our dataset. Ligands crystallized in similar proteins and highly ligand-efficient compounds with five or fewer rotatable bonds were less likely to adjust conformation when binding. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Dual Functional Small Molecule Probes as Fluorophore and Ligand for Misfolding Proteins

    OpenAIRE

    Zhang, Xueli; Ran, Chongzhao

    2013-01-01

    Misfolding of a protein is a destructive process for variety of diseases that include neurodegenerative diseases such as Alzheimer’s disease, Parkinson disease, Huntington disease, mad cow disease, amyotrophic lateral sclerosis (ALS), and frontal temporal dementia (FTD), and other non-CNS diseases such as diabetes, cystic fibrosis, and lysosomal storage diseases. Formation of various misfunctional large assembles of the misfolded protein is the primary consequence. To detect the formation of ...

  19. Binary classification of protein molecules into intrinsically disordered and ordered segments

    Directory of Open Access Journals (Sweden)

    Gojobori Takashi

    2011-06-01

    Full Text Available Abstract Background Although structural domains in proteins (SDs are important, half of the regions in the human proteome are currently left with no SD assignments. These unassigned regions consist not only of novel SDs, but also of intrinsically disordered (ID regions since proteins, especially those in eukaryotes, generally contain a significant fraction of ID regions. As ID regions can be inferred from amino acid sequences, a method that combines SD and ID region assignments can determine the fractions of SDs and ID regions in any proteome. Results In contrast to other available ID prediction programs that merely identify likely ID regions, the DICHOT system we previously developed classifies the entire protein sequence into SDs and ID regions. Application of DICHOT to the human proteome revealed that residue-wise ID regions constitute 35%, SDs with similarity to PDB structures comprise 52%, while SDs with no similarity to PDB structures account for the remaining 13%. The last group consists of novel structural domains, termed cryptic domains, which serve as good targets of structural genomics. The DICHOT method applied to the proteomes of other model organisms indicated that eukaryotes generally have high ID contents, while prokaryotes do not. In human proteins, ID contents differ among subcellular localizations: nuclear proteins had the highest residue-wise ID fraction (47%, while mitochondrial proteins exhibited the lowest (13%. Phosphorylation and O-linked glycosylation sites were found to be located preferentially in ID regions. As O-linked glycans are attached to residues in the extracellular regions of proteins, the modification is likely to protect the ID regions from proteolytic cleavage in the extracellular environment. Alternative splicing events tend to occur more frequently in ID regions. We interpret this as evidence that natural selection is operating at the protein level in alternative splicing. Conclusions We classified

  20. The chemical component dictionary: complete descriptions of constituent molecules in experimentally determined 3D macromolecules in the Protein Data Bank.

    Science.gov (United States)

    Westbrook, John D; Shao, Chenghua; Feng, Zukang; Zhuravleva, Marina; Velankar, Sameer; Young, Jasmine

    2015-04-15

    The Chemical Component Dictionary (CCD) is a chemical reference data resource that describes all residue and small molecule components found in Protein Data Bank (PDB) entries. The CCD contains detailed chemical descriptions for standard and modified amino acids/nucleotides, small molecule ligands and solvent molecules. Each chemical definition includes descriptions of chemical properties such as stereochemical assignments, chemical descriptors, systematic chemical names and idealized coordinates. The content, preparation, validation and distribution of this CCD chemical reference dataset are described. The CCD is updated regularly in conjunction with the scheduled weekly release of new PDB structure data. The CCD and amino acid variant reference datasets are hosted in the public PDB ftp repository at ftp://ftp.wwpdb.org/pub/pdb/data/monomers/components.cif.gz, ftp://ftp.wwpdb.org/pub/pdb/data/monomers/aa-variants-v1.cif.gz, and its mirror sites, and can be accessed from http://wwpdb.org. jwest@rcsb.rutgers.edu. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Tamm–Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System

    Directory of Open Access Journals (Sweden)

    Tsai-Hung Wu

    2018-01-01

    Full Text Available Tamm–Horsfall protein (THP, or uromodulin (UMOD, is an 80–90-kDa phosphatidylinositol-anchored glycoprotein produced exclusively by the renal tubular cells in the thick ascending limb of the loop of Henle. Physiologically, THP is implicated in renal countercurrent gradient formation, sodium homeostasis, blood pressure regulation, and a defense molecule against infections in the urinary system. Investigations have also revealed that THP is an effective binding ligand for serum albumin, immunoglobulin G light chains, complement components C1 and C1q, interleukin (IL-1β, IL-6, IL-8, tumor necrosis factor (TNF-α, and interferon-γ through its carbohydrate side chains for maintaining circulatory and renal immune homeostasis. Thus, THP can be regarded as part of the innate immune system. UMOD mutations play crucial roles in congenital urolithiasis, hereditary hyperuricemia/gout, and medullary cystic kidney diseases. Recent investigations have focused on the immunomodulatory effects of THP on immune cells and on THP as a disease biomarker of acute and chronic kidney diseases. Our studies have suggested that normal urinary THP, through its epidermal growth factor (EGF-like domains, binds to the surface-expressed EGF-like receptors, cathepsin G, or lactoferrin to enhance polymorphonuclear leukocyte phagocytosis, proinflammatory cytokine production by monocytes/macrophages, and lymphocyte proliferation by activating the Rho family and mitogen-activated protein kinase signaling pathways. Furthermore, our data support both an intact protein core structure and carbohydrate side chains are important for the different protein-binding capacities of THP. Prospectively, parts of the whole THP molecule may be used for anti-TNF-α therapy in inflammatory diseases, autoantibody-depleting therapy in autoimmune disorders, and immune intensification in immunocompromised hosts.

  2. Population of nitrogen molecule electron states and structure of the fast ionization wave

    CERN Document Server

    Pancheshnyi, S V; Starikovskii, A Y

    1999-01-01

    The excitation of N sub 2 (C sup 3 supPI sub u , nu=0) and N sup + sub 2 (B sup 2 supSIGMA sup + sub u , nu=0) electron states has been studied by using a time-resolved emission spectroscopy technique. To excite the above states, the nanosecond, high-voltage, periodic impulsed discharge at low pressures in the form of the fast ionization wave (FIW) was used. The electron concentration and the average energy, electric field were found on the basis of experimental data. The spacial-temporal structure of the FIW front was investigated. It has been shown that the generation of the required electron concentration, as well as the electron level population take place behind the FIW front in residual fields. Sections corresponding to the 'electric' and 'luminous' FIW fronts are essentially separated in space. The proposed modelling electron energy distribution function describes qualitatively general regularities of the breakdown propagation in the whole range of parameters under study. (author)

  3. Using the multi-objective optimization replica exchange Monte Carlo enhanced sampling method for protein-small molecule docking.

    Science.gov (United States)

    Wang, Hongrui; Liu, Hongwei; Cai, Leixin; Wang, Caixia; Lv, Qiang

    2017-07-10

    In this study, we extended the replica exchange Monte Carlo (REMC) sampling method to protein-small molecule docking conformational prediction using RosettaLigand. In contrast to the traditional Monte Carlo (MC) and REMC sampling methods, these methods use multi-objective optimization Pareto front information to facilitate the selection of replicas for exchange. The Pareto front information generated to select lower energy conformations as representative conformation structure replicas can facilitate the convergence of the available conformational space, including available near-native structures. Furthermore, our approach directly provides min-min scenario Pareto optimal solutions, as well as a hybrid of the min-min and max-min scenario Pareto optimal solutions with lower energy conformations for use as structure templates in the REMC sampling method. These methods were validated based on a thorough analysis of a benchmark data set containing 16 benchmark test cases. An in-depth comparison between MC, REMC, multi-objective optimization-REMC (MO-REMC), and hybrid MO-REMC (HMO-REMC) sampling methods was performed to illustrate the differences between the four conformational search strategies. Our findings demonstrate that the MO-REMC and HMO-REMC conformational sampling methods are powerful approaches for obtaining protein-small molecule docking conformational predictions based on the binding energy of complexes in RosettaLigand.

  4. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

    International Nuclear Information System (INIS)

    Mattila, Elina; Marttila, Heidi; Sahlberg, Niko; Kohonen, Pekka; Tähtinen, Siri; Halonen, Pasi; Perälä, Merja; Ivaska, Johanna

    2010-01-01

    T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening

  5. Protein logic: a statistical mechanical study of signal integration at the single-molecule level.

    Science.gov (United States)

    de Ronde, Wiet; Rein ten Wolde, Pieter; Mugler, Andrew

    2012-09-05

    Information processing and decision-making is based upon logic operations, which in cellular networks has been well characterized at the level of transcription. In recent years, however, both experimentalists and theorists have begun to appreciate that cellular decision-making can also be performed at the level of a single protein, giving rise to the notion of protein logic. Here we systematically explore protein logic using a well-known statistical mechanical model. As an example system, we focus on receptors that bind either one or two ligands, and their associated dimers. Notably, we find that a single heterodimer can realize any of the 16 possible logic gates, including the XOR gate, by variation of biochemical parameters. We then introduce what to our knowledge is a novel idea: that a set of receptors with fixed parameters can encode functionally unique logic gates simply by forming different dimeric combinations. An exhaustive search reveals that the simplest set of receptors (two single-ligand receptors and one double-ligand receptor) can realize several different groups of three unique gates, a result for which the parametric analysis of single receptors and dimers provides a clear interpretation. Both results underscore the surprising functional freedom readily available to cells at the single-protein level. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...

  7. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy

  8. Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

    Science.gov (United States)

    Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

    2015-05-01

    Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

  9. High resolution mass spectrometry method and system for analysis of whole proteins and other large molecules

    Science.gov (United States)

    Reilly, Peter T. A. [Knoxville, TN; Harris, William A [Naperville, IL

    2010-03-02

    A matrix assisted laser desorption/ionization (MALDI) method and related system for analyzing high molecular weight analytes includes the steps of providing at least one matrix-containing particle inside an ion trap, wherein at least one high molecular weight analyte molecule is provided within the matrix-containing particle, and MALDI on the high molecular weight particle while within the ion trap. A laser power used for ionization is sufficient to completely vaporize the particle and form at least one high molecular weight analyte ion, but is low enough to avoid fragmenting the high molecular weight analyte ion. The high molecular weight analyte ion is extracted out from the ion trap, and is then analyzed using a detector. The detector is preferably a pyrolyzing and ionizing detector.

  10. Biosynthetic incorporation of telluromethionine into dihydrofolate reductase and crystallographic analysis of the distribution of tellurium atoms in the protein molecule

    Energy Technology Data Exchange (ETDEWEB)

    Kunkle, M.G.; Lewinski, K.; Boles, J.O.; Dunlap, R.B.; Odom, J.D.; Lebioda, L. [Univ. of South Carolina, Columbia, SC (United States)

    1994-12-01

    Recent successes in crystallographic studies of proteins with methionine (Met) residues replaced with SeMet, pioneered by Hendrickson and coworkers, inspired us to replace Met with TeMet in Escherichia coli dihydrofolate reductase (DHFR). E. coli DHFR, which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, consists of 159 residues, 5 of which are Met. TeMet was incorporated into DHFR using the Met auxotroph, E. coli DL41, carrying the expression vector pWT8 with an IPTG inducible promoter and ampicillin resistance gene. The enzyme was purified by successive chromatography on Q-Sepharose and PHenyl Sepharose resins, yielding milligram quantities of homogeneous enzyme with a specific activity of 40 units/mg. TeMet DHFR exhibits kinetic properties similar to those of wt DHFR. Amino acid analysis indicated 3 authentic Met residues in TeMet DHFR, whereas atomic absorption spectroscopy detected 2 Te per protein molecule. Amino acid sequence analysis results suggested that only authentic Met was present in the first three Met positions (1,16,and 20). Crystals of Te-DHFR were grown in the presence of methotrexate from PEG 4000 and were isomorphous with wt-DHFR crystals grown from ethanol. Difference Fourier maps and restrained least-squares refinement show very little, if any, Te in the first three Met positions: Met{sup 1}, Met{sup 16}, and Met{sup 20}, whereas the occupancy of Te in positions 42 and 92 is 0.64. Apparently, the process of folding, subsequent purification, and crystallization select DHFR molecules with Te in Met{sup 42} and Met{sup 92}. Replacing Met with TeMet provides an internal probe that should facilitate structural and mechanistic studies of proteins.

  11. Hydrogen/deuterium isotope effects in water and aqueous solutions of organic molecules and proteins

    International Nuclear Information System (INIS)

    Price, David L.; Fu, Ling; Bermejo, F. Javier; Fernandez-Alonso, Felix; Saboungi, Marie-Louise

    2013-01-01

    Highlights: ► Hydrogen/deuterium substitution has significant effects in hydrogenous materials. ► The effects can involve structure, phase behavior and protein stability. ► The effects must be kept in mind in the interpretation of scattering experiments. ► The effects may be mitigated by an appropriate choice of experimental conditions. - Abstract: It is pointed out that hydrogen/deuterium substitution, frequently used in neutron scattering studies of the structure and dynamics of hydrogenous samples, can have significant effects on structure, phase behavior and protein stability. The effects must be kept in mind in the interpretation of such experiments. In suitable cases, these effects can be mitigated by an appropriate choice of experimental conditions

  12. The application of polythiol molecules for protein immobilisation on sensor surfaces.

    Science.gov (United States)

    Kyprianou, Dimitris; Guerreiro, Antonio R; Nirschl, Martin; Chianella, Iva; Subrahmanyam, Sreenath; Turner, Anthony P F; Piletsky, Sergey

    2010-01-15

    The immobilisation of bio-receptors on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The development of a protocol for biomolecule immobilisation onto a surface plasmon resonance (SPR) sensor surface using inexpensive polythiol compounds is presented here. The method used here is based on the reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. The self-assembled thiol monolayers were characterised using contact angle and XPS. The possibility to immobilise proteins on monolayers was assessed by employing BSA as a model protein. For the polythiol layers exhibiting the best performance, a general protocol was optimised suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti Salmonella typhimurium. The kinetic data was obtained for PSA binding to anti-PSA and for S. typhimurium cells with a detection limit of 5x10(6) cells mL(-1) with minimal non-specific binding of other biomolecules. These findings make this technique a very promising alternative for amine coupling compared to peptide bond formation. Additionally, it offers opportunity for immobilising proteins (even those with low isoelectric point) on neutral polythiol layers without any activation step. Copyright 2009 Elsevier B.V. All rights reserved.

  13. Docking ligands into flexible and solvated macromolecules. 7. Impact of protein flexibility and water molecules on docking-based virtual screening accuracy.

    Science.gov (United States)

    Therrien, Eric; Weill, Nathanael; Tomberg, Anna; Corbeil, Christopher R; Lee, Devin; Moitessier, Nicolas

    2014-11-24

    The use of predictive computational methods in the drug discovery process is in a state of continual growth. Over the last two decades, an increasingly large number of docking tools have been developed to identify hits or optimize lead molecules through in-silico screening of chemical libraries to proteins. In recent years, the focus has been on implementing protein flexibility and water molecules. Our efforts led to the development of Fitted first reported in 2007 and further developed since then. In this study, we wished to evaluate the impact of protein flexibility and occurrence of water molecules on the accuracy of the Fitted docking program to discriminate active compounds from inactive compounds in virtual screening (VS) campaigns. For this purpose, a total of 171 proteins cocrystallized with small molecules representing 40 unique enzymes and receptors as well as sets of known ligands and decoys were selected from the Protein Data Bank (PDB) and the Directory of Useful Decoys (DUD), respectively. This study revealed that implementing displaceable crystallographic or computationally placed particle water molecules and protein flexibility can improve the enrichment in active compounds. In addition, an informed decision based on library diversity or research objectives (hit discovery vs lead optimization) on which implementation to use may lead to significant improvements.

  14. Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

    Science.gov (United States)

    Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

    2011-03-01

    Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

  15. Computing Rates of Small Molecule Diffusion Through Protein Channels Using Markovian Milestoning

    Science.gov (United States)

    Abrams, Cameron

    2014-03-01

    Measuring diffusion rates of ligands plays a key role in understanding the kinetic processes inside proteins. For example, although many molecular simulation studies have reported free energy barriers to infer rates for CO diffusion in myoglobin (Mb), they typically do not include direct calculation of diffusion rates because of the long simulation times needed to infer these rates with statistical accuracy. We show in this talk how to apply Markovian milestoning along minimum free-energy pathways to calculate diffusion rates of CO inside Mb. In Markovian milestoning, one partitions a suitable reaction coordinate space into regions and performs restrained molecular dynamics in each region to accumulate kinetic statistics that, when assembled across regions, provides an estimate of the mean first-passage time between states. The mean escape time for CO directly from the so-called distal pocket (DP) through the histidine gate (HG) is estimated at about 24 ns, confirming the importance of this portal for CO. But Mb is known to contain several internal cavities, and cavity-to-cavity diffusion rates are also computed and used to build a complete kinetic network as a Markov state model. Within this framework, the effective mean time of escape to the solvent through HG increases to 30 ns. Our results suggest that carrier protein structure may have evolved under pressure to modulate dissolved gas release rates using a network of ligand-accessible cavities. Support: NIH R01GM100472.

  16. Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-05-01

    Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

  17. Evaluation of EML4-ALK Fusion Proteins in Non–Small Cell Lung Cancer Using Small Molecule Inhibitors

    Directory of Open Access Journals (Sweden)

    Yongjun Li

    2011-01-01

    Full Text Available The echinoderm microtubule–associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non–small cell lung cancer and is mu-tually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non–small cell lung cancer (NSCLC. We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mech-anism of EML4-ALK inhibition by a small molecule inhibitor.

  18. Chitinase-3-like Protein 1: A Progranulin Downstream Molecule and Potential Biomarker for Gaucher Disease

    Directory of Open Access Journals (Sweden)

    Jinlong Jian

    2018-02-01

    Full Text Available We recently reported that progranulin (PGRN is a novel regulator of glucocerebrosidase and its deficiency associates with Gaucher Diseases (GD (Jian et al., 2016a; Jian et al., 2018. To isolate the relevant downstream molecules, we performed a whole genome microarray and mass spectrometry analysis, which led to the isolation of Chitinase-3-like-1 (CHI3L1 as one of the up-regulated genes in PGRN null mice. Elevated levels of CHI3L1 were confirmed by immunoblotting and immunohistochemistry. In contrast, treatment with recombinant Pcgin, a derivative of PGRN, as well as imigluerase, significantly reduced the expressions of CHI3L1 in both PGRN null GD model and the fibroblasts from GD patients. Serum levels of CHIT1, a clinical biomarker for GD, were significantly higher in GD patients than healthy controls (51.16 ± 2.824 ng/ml vs 35.07 ± 2.099 ng/ml, p < 0.001. Similar to CHIT1, serum CHI3L1 was also significantly increased in GD patients compared with healthy controls (1736 ± 152.1 pg/ml vs 684.7 ± 68.20 pg/ml, p < 0.001. Whereas the PGRN level is significantly reduced in GD patients as compared to the healthy control (91.56 ± 3.986 ng/ml vs 150.6 ± 4.501, p < 0.001. Collectively, these results indicate that CHI3L1 may be a previously unrecognized biomarker for diagnosing GD and for evaluating the therapeutic effects of new GD drug(s.

  19. Molecular architecture of a complex between an adhesion protein from the malaria parasite and intracellular adhesion molecule 1

    DEFF Research Database (Denmark)

    Brown, Alan; Turner, Louise; Christoffersen, Stig

    2013-01-01

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria....... The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from......, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLß domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1...

  20. What precision-protein-tuning and nano-resolved single molecule sciences can do for each other.

    Science.gov (United States)

    Milles, Sigrid; Lemke, Edward A

    2013-01-01

    While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. Copyright © 2013 WILEY Periodicals, Inc.

  1. Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors.

    Science.gov (United States)

    Jayakumar, Jayanthi; Anishetty, Sharmila

    2014-05-01

    Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Do vulnerable populations consume adequate amounts of dietary protein?

    Science.gov (United States)

    In the previous year there has been a renewed interest in the adequacy of protein intake to maintain optimal health and to promote normal growth and development (1, 2). In this issue of the Journal there is an excellent report on protein consumption among children aged 6–36 mo from low-income countr...

  3. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Science.gov (United States)

    Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L T O

    2011-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  4. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Directory of Open Access Journals (Sweden)

    Rosane Oliveira

    Full Text Available Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30 and LIC12238. We have employed Escherichia coli BL21 (SI strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively. In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa. Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  5. Genetic polymorphism of blood proteins in a population of shetland ponies

    NARCIS (Netherlands)

    Buis, R.C.

    1976-01-01

    Genetic variation of proteins (protein polymorphism) is widespread among many animal species. The biological significance of protein polymorphism has been the subject of many studies. This variation has a supporting function for population genetic studies as a source of genetic markers. In

  6. Sources of dietary protein and risk of hypertension in a general Dutch population

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.; Engberink, M.F.; Geleijnse, J.M.; Boer, J.M.A.; Verschuren, W.M.M.

    2012-01-01

    Evidence suggests a small beneficial effect of dietary protein on blood pressure (BP), especially for plant protein. We examined the relationship between several types of dietary protein (total, plant, animal, dairy, meat and grain) and the risk of hypertension in a general population of 3588 Dutch

  7. Radiolabelled molecules for imaging the translocator protein (18 kDa) using positron emission tomography

    International Nuclear Information System (INIS)

    Dolle, F.; Luus, C.; Reynolds, A.; Kassiou, M.

    2009-01-01

    The translocator protein (18 kDa) (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR), was originally identified as an alternate binding site for the central benzodiazepine receptor (CBR) ligand, diazepam, in the periphery, but has now been distinguished as a novel site. The TSPO is ubiquitously expressed in peripheral tissues but only minimally in the healthy brain and increased levels of TSPO expression have been noted in neuro inflammatory conditions such as Alzheimer's disease, Parkinson's disease and stroke. This increase in TSPO expression has been reported to coincide with the process of micro-glial activation, whereby the brain's intrinsic immune system becomes active. Therefore, by using recently developed high affinity, selective TSPO ligands in conjunction with functional imaging modalities such as positron emission tomography (PET), it becomes possible to study the process of micro-glial activation in the living brain. A number of high affinity ligands, the majority of which are C, N-substituted acetamide derivatives, have been successfully radiolabelled and used in in vivo studies of the TSPO and the process of micro-glial activation. This review highlights recent achievements (up to December 2008) in the field of functional imaging of the TSPO as well as the radio-syntheses involved in such studies. (authors)

  8. Single-molecule spectroscopy of LHCSR1 protein dynamics identifies two distinct states responsible for multi-timescale photosynthetic photoprotection

    Science.gov (United States)

    Kondo, Toru; Pinnola, Alberta; Chen, Wei Jia; Dall'Osto, Luca; Bassi, Roberto; Schlau-Cohen, Gabriela S.

    2017-08-01

    In oxygenic photosynthesis, light harvesting is regulated to safely dissipate excess energy and prevent the formation of harmful photoproducts. Regulation is known to be necessary for fitness, but the molecular mechanisms are not understood. One challenge has been that ensemble experiments average over active and dissipative behaviours, preventing identification of distinct states. Here, we use single-molecule spectroscopy to uncover the photoprotective states and dynamics of the light-harvesting complex stress-related 1 (LHCSR1) protein, which is responsible for dissipation in green algae and moss. We discover the existence of two dissipative states. We find that one of these states is activated by pH and the other by carotenoid composition, and that distinct protein dynamics regulate these states. Together, these two states enable the organism to respond to two types of intermittency in solar intensity—step changes (clouds and shadows) and ramp changes (sunrise), respectively. Our findings reveal key control mechanisms underlying photoprotective dissipation, with implications for increasing biomass yields and developing robust solar energy devices.

  9. Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

    Science.gov (United States)

    Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

    2007-01-01

    Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores. Copyright (c) 2007 John Wiley & Sons, Ltd.

  10. Toward Small-Molecule Inhibition of Protein-Protein Interactions: General Aspects and Recent Progress in Targeting Costimulatory and Coinhibitory (Immune Checkpoint) Interactions.

    Science.gov (United States)

    Bojadzic, Damir; Buchwald, Peter

    2018-05-30

    Protein-protein interactions (PPIs) that are part of the costimulatory and coinhibitory (immune checkpoint) signaling are critical for adequate T cell response and are important therapeutic targets for immunomodulation. Biologics targeting them have already achieved considerable clinical success in the treatment of autoimmune diseases or transplant recipients (e.g., abatacept, belatacept, and belimumab) as well as cancer (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab). In view of such progress, there have been only relatively limited efforts toward developing small-molecule PPI inhibitors (SMPPIIs) targeting these cosignaling interactions, possibly because they, as all other PPIs, are difficult to target by small molecules and were not considered druggable. Nevertheless, substantial progress has been achieved during the last decade. SMPPIIs proving the feasibility of such approaches have been identified through various strategies for a number of cosignaling interactions including CD40-CD40L, OX40-OX40L, BAFFR-BAFF, CD80-CD28, and PD-1-PD-L1s. Here, after an overview of the general aspects and challenges of SMPPII-focused drug discovery, we review them briefly together with relevant structural, immune-signaling, physicochemical, and medicinal chemistry aspects. While so far only a few of these SMPPIIs have shown activity in animal models (DRI-C21045 for CD40-D40L, KR33426 for BAFFR-BAFF) or reached clinical development (RhuDex for CD80-CD28, CA-170 for PD-1-PD-L1), there is proof-of-principle evidence for the feasibility of such approaches in immunomodulation. They can result in products that are easier to develop/manufacture and are less likely to be immunogenic or encounter postmarket safety events than corresponding biologics, and, contrary to them, can even become orally bioavailable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. 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.

  12. Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

    Science.gov (United States)

    Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

    2009-04-01

    The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

  13. Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

    Science.gov (United States)

    Myre, Michael A.; Washicosky, Kevin; Moir, Robert D.; Tesco, Giuseppina; Tanzi, Rudolph E.; Wasco, Wilma

    2013-01-01

    The detection of cell cycle proteins in Alzheimer’s disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

  14. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  15. Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population.

    Science.gov (United States)

    Micera, Alessandra; Quaranta, Luciano; Esposito, Graziana; Floriani, Irene; Pocobelli, Augusto; Saccà, Sergio Claudio; Riva, Ivano; Manni, Gianluca; Oddone, Francesco

    2016-02-01

    Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

  16. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population

    DEFF Research Database (Denmark)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine

    2014-01-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum...... proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type....... Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations...

  17. Theoretical study of the influence of intense femtosecond laser field on the evolution of the wave packet and the population of NaRb molecule

    International Nuclear Information System (INIS)

    Ning, Ma; Mei-Shan, Wang; Chuan-Lu, Yang; Xiao-Guang, Ma; De-Hua, Wang

    2010-01-01

    Employing the two-state model and the time-dependent wave packet method, we have investigated the influences of the parameters of the intense femtosecond laser field on the evolution of the wave packet, as well as the population of ground and double-minimum electronic states of the NaRb molecule. For the different laser wavelengths, the evolution of the wave packet of 6 1 σ + state with time and internuclear distance is different, and the different laser intensity brings different influences on the population of the electronic states of the NaRb molecule. One can control the evolutions of wave packet and the population in each state by varying the laser parameters appropriately, which will be a benefit for the light manipulation of atomic and molecular processes. (atomic and molecular physics)

  18. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    Science.gov (United States)

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  19. Expression of neural cell adhesion molecules and neurofilament protein isoforms in Ewing's sarcoma of bone and soft tissue sarcomas of other than rhabdomyosarcoma

    NARCIS (Netherlands)

    Molenaar, W.M.; Muntinghe, F.L.H.

    1999-01-01

    In a previous study, it was shown that rhabdomyosarcomas widely express "neural" markers, such as neural cell adhesion molecules (N-CAM) and neurofilament protein isoforms, In the current study, a series of Ewing's sarcomas of bone and soft tissue sarcomas other than rhabdomyosarcoma was probed for

  20. Conservative, unconditionally stable discretization methods for Hamiltonian equations, applied to wave motion in lattice equations modeling protein molecules

    Science.gov (United States)

    LeMesurier, Brenton

    2012-01-01

    A new approach is described for generating exactly energy-momentum conserving time discretizations for a wide class of Hamiltonian systems of DEs with quadratic momenta, including mechanical systems with central forces; it is well-suited in particular to the large systems that arise in both spatial discretizations of nonlinear wave equations and lattice equations such as the Davydov System modeling energetic pulse propagation in protein molecules. The method is unconditionally stable, making it well-suited to equations of broadly “Discrete NLS form”, including many arising in nonlinear optics. Key features of the resulting discretizations are exact conservation of both the Hamiltonian and quadratic conserved quantities related to continuous linear symmetries, preservation of time reversal symmetry, unconditional stability, and respecting the linearity of certain terms. The last feature allows a simple, efficient iterative solution of the resulting nonlinear algebraic systems that retain unconditional stability, avoiding the need for full Newton-type solvers. One distinction from earlier work on conservative discretizations is a new and more straightforward nearly canonical procedure for constructing the discretizations, based on a “discrete gradient calculus with product rule” that mimics the essential properties of partial derivatives. This numerical method is then used to study the Davydov system, revealing that previously conjectured continuum limit approximations by NLS do not hold, but that sech-like pulses related to NLS solitons can nevertheless sometimes arise.

  1. Immunoassay of 5-methyltetrahydrofolate: use of 125I-labeled protein A as the tracer molecule for specific antibody

    International Nuclear Information System (INIS)

    Langone, J.J.

    1980-01-01

    A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. 125 I-Labeled staphylococcal Protein A ( 125 I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of 125 I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific; 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15 to 20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits

  2. Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

    Science.gov (United States)

    Shen, Jia; James, Aaron W.; Zhang, Xinli; Pang, Shen; Zara, Janette N.; Asatrian, Greg; Chiang, Michael; Lee, Min; Khadarian, Kevork; Nguyen, Alan; Lee, Kevin S.; Siu, Ronald K.; Tetradis, Sotirios; Ting, Kang; Soo, Chia

    2017-01-01

    The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. PMID:26772960

  3. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

    Science.gov (United States)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

    2014-09-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

  4. Modulation of Calcium Oxalate Crystallization by Proteins and Small Molecules Investigated by In Situ Atomic Force Microscopy

    Science.gov (United States)

    Qiu, R.; Orme, C.; Cody, A. M.; Wierzbicki, A.; Hoyer, J.; Nancollas, G.; de Yoreo, J.

    2002-12-01

    Understanding the physical mechanisms by which biological inhibitors control nucleation and growth of inorganic crystals is a major focus of biomineral research. Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is also a source of pathogenesis in humans where it causes kidney stone disease. Although a great deal of research has been carried out on the modulation COM by proteins and small molecules, the basic mechanism has not yet been understood. However, because the proteins that play a role in COM growth have been identified and sequenced, COM provides an excellent model system for research into biomineral growth. In this study, in situ atomic force microscopy (AFM) was used to monitor the COM surface under controlled growth conditions both from pure solutions and those doped with citrate and osteopontin (OPN) in order to determine their effects on surface morphology and growth dynamics at the molecular level. As with other solution-grown crystals such as calcite, COM grows on complex dislocation hillocks. In pure solution, while growth on the (010) face is isotropic, hillocks on the (-101) face exhibit anisotropic step kinetics. Steps of [-10-1] and orientation are clearly delineated with the [-10-1] being the fast growing direction. When citrate is added to the solution, both growth rate and morphology are drastically changed on (-101) face, especially along the [-10-1] direction. This results in isotropic disc-shaped hillocks a shape that is then reflected in the macroscopic growth habit. In contrast, no large growth changes were observed on the (010) facet. At the same time, molecular modeling predicts an excellent fit of the citrate ion into the (-101) plane and a poor fit to the (010) face. Here we propose a model that reconciles the step-specific interactions implied by the AFM results with the face-specific predictions of the calculations. Finally, we present the results of doping with aspartic acid as well as OPN, an

  5. Detecting differential protein expression in large-scale population proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Soyoung; Qian, Weijun; Camp, David G.; Smith, Richard D.; Tompkins, Ronald G.; Davis, Ronald W.; Xiao, Wenzhong

    2014-06-17

    Mass spectrometry-based high-throughput quantitative proteomics shows great potential in clinical biomarker studies, identifying and quantifying thousands of proteins in biological samples. However, methods are needed to appropriately handle issues/challenges unique to mass spectrometry data in order to detect as many biomarker proteins as possible. One issue is that different mass spectrometry experiments generate quite different total numbers of quantified peptides, which can result in more missing peptide abundances in an experiment with a smaller total number of quantified peptides. Another issue is that the quantification of peptides is sometimes absent, especially for less abundant peptides and such missing values contain the information about the peptide abundance. Here, we propose a Significance Analysis for Large-scale Proteomics Studies (SALPS) that handles missing peptide intensity values caused by the two mechanisms mentioned above. Our model has a robust performance in both simulated data and proteomics data from a large clinical study. Because varying patients’ sample qualities and deviating instrument performances are not avoidable for clinical studies performed over the course of several years, we believe that our approach will be useful to analyze large-scale clinical proteomics data.

  6. Population distribution of flexible molecules from maximum entropy analysis using different priors as background information: application to the Φ, Ψ-conformational space of the α-(1-->2)-linked mannose disaccharide present in N- and O-linked glycoproteins.

    Science.gov (United States)

    Säwén, Elin; Massad, Tariq; Landersjö, Clas; Damberg, Peter; Widmalm, Göran

    2010-08-21

    The conformational space available to the flexible molecule α-D-Manp-(1-->2)-α-D-Manp-OMe, a model for the α-(1-->2)-linked mannose disaccharide in N- or O-linked glycoproteins, is determined using experimental data and molecular simulation combined with a maximum entropy approach that leads to a converged population distribution utilizing different input information. A database survey of the Protein Data Bank where structures having the constituent disaccharide were retrieved resulted in an ensemble with >200 structures. Subsequent filtering removed erroneous structures and gave the database (DB) ensemble having three classes of mannose-containing compounds, viz., N- and O-linked structures, and ligands to proteins. A molecular dynamics (MD) simulation of the disaccharide revealed a two-state equilibrium with a major and a minor conformational state, i.e., the MD ensemble. These two different conformation ensembles of the disaccharide were compared to measured experimental spectroscopic data for the molecule in water solution. However, neither of the two populations were compatible with experimental data from optical rotation, NMR (1)H,(1)H cross-relaxation rates as well as homo- and heteronuclear (3)J couplings. The conformational distributions were subsequently used as background information to generate priors that were used in a maximum entropy analysis. The resulting posteriors, i.e., the population distributions after the application of the maximum entropy analysis, still showed notable deviations that were not anticipated based on the prior information. Therefore, reparameterization of homo- and heteronuclear Karplus relationships for the glycosidic torsion angles Φ and Ψ were carried out in which the importance of electronegative substituents on the coupling pathway was deemed essential resulting in four derived equations, two (3)J(COCC) and two (3)J(COCH) being different for the Φ and Ψ torsions, respectively. These Karplus relationships are denoted

  7. Control of population of excited nitrogen molecules by mixing hydrogen in low pressure discharge; Chisso jun`antei reiki bunshi mitsudo no quenching ni yoru seigyo no kento

    Energy Technology Data Exchange (ETDEWEB)

    Uematsu, K.; Yumoto, M.; Sakai, T. [Musashi Institute of Technology, Tokyo (Japan)

    1998-06-01

    The authors have studied on surface treatment of PTFE by a low pressure discharge. It is deduced that excited nitrogen molecules contribute to introduce polar components on the surface. To confirm the speculation, we tried to change population of metastable nitrogen N2 (A{sup 3}{Sigma}u{sup +}) by quenching precursor N2 (B{sup 3}{pi}g), with hydrogen molecule. The decrease of relaxation time which indicates a change of excited molecule and measured by emission spectroscopy using a time after glow method was obtained. As a result, the relaxation times of N2 (B{sup 3}{pi}g) and N2 (A{sup 3}{Sigma}u{sup +}) decreased to 55% and 20% respectively, when mixing ratio of hydrogen was 3%. It was also deduced that hydrogen atom may take a part in a quenching process of N2 (A{sup 3}{Sigma}u{sup +}). 14 refs., 11 figs., 1 tab.

  8. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    International Nuclear Information System (INIS)

    Poortmans, J.R.; Carpentier, A.; Pereira-Lancha, L.O.; Lancha, A. Jr.

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ( 13 C-lysine, 15 N-glycine, 2 H 5 -phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg −1 ·day −1 compared to 0.8 g·kg −1 ·day −1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h

  9. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Energy Technology Data Exchange (ETDEWEB)

    Poortmans, J.R.; Carpentier, A. [Laboratory for Biometry and Sport Nutrition, Faculty of Motor Sciences, Free University of Brussels, Brussels (Belgium); Pereira-Lancha, L.O. [Departamento de Nutrição, Instituto Vita, São Paulo, SP (Brazil); Lancha, A. Jr. [Laboratório de Nutrição Aplicada à Atividade Motora, Escola de Educação Física e Esporte, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-08

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ({sup 13}C-lysine, {sup 15}N-glycine, {sup 2}H{sub 5}-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg{sup −1}·day{sup −1} compared to 0.8 g·kg{sup −1}·day{sup −1} in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  10. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Directory of Open Access Journals (Sweden)

    J.R. Poortmans

    2012-10-01

    Full Text Available Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes compared to resistance exercise, which induces fiber hypertrophy (myofibrils. Nitrogen balance (difference between protein intake and protein degradation for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins and carbohydrate (± 30 g maltodextrine drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  11. The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

    Directory of Open Access Journals (Sweden)

    Stéphanie eCottier

    2011-12-01

    Full Text Available Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx. In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA binding domain (encoded in the yeast strain, and the bioactive molecule part binding to its potential protein target fused to a DNA activating domain (encoded on a cDNA expression vector. During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discussed the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

  12. Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor.

    Science.gov (United States)

    Knight, Martha; Finn, Thomas M; Zehmer, John; Clayton, Adam; Pilon, Aprile

    2011-09-09

    An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB

    Science.gov (United States)

    Zhang, Yanling.; Zhen, Wei.; Maechler, Pierre; Liu, Dongmin

    2013-01-01

    Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of T2D. Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis, and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of anti-apoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and PDX-1 expression. Chronic hyperlipidemia significantly diminished cAMP production, PKA activation, and CREB phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48 h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol–stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade. PMID:22819546

  14. Organic conjugated small molecule materials based optical probe for rapid, colorimetric and UV-vis spectral detection of phosphorylated protein in placental tissue.

    Science.gov (United States)

    Wang, Yanfang; Yang, Na; Liu, Yi

    2018-04-05

    A novel organic small molecule with D-Pi-A structure was prepared, which was found to be a promising colorimetric and ratiometric UV-vis spetral probe for detection of phosphorylated proteins with the help of tetravalent zirconium ion. Such optical probe based on chromophore WYF-1 shows a rapid response (within 10s) and high selectivity and sensitivity for phosphorylated proteins, giving distinct colorimetric and ratiometric UV-vis changes at 720 and 560nm. The detection limit for phosphorylated proteins was estimated to be 100nM. In addition, detection of phosphorylated proteins in placental tissue samples with this probe was successfully applied, which indicates that this probe holds great potential for phosphorylated proteins detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  16. SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

    Science.gov (United States)

    Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

    2009-07-01

    Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

  17. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer.

    Science.gov (United States)

    Wollman, Adam J M; Miller, Helen; Zhou, Zhaokun; Leake, Mark C

    2015-04-01

    DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

  18. Amino acid sequences mediating vascular cell adhesion molecule 1 binding to integrin alpha 4: homologous DSP sequence found for JC polyoma VP1 coat protein

    Directory of Open Access Journals (Sweden)

    Michael Andrew Meyer

    2013-07-01

    Full Text Available The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4 to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3. For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

  19. Immunological network activation by low-dose rate irradiation. Analysis of cell populations and cell surface molecules in whole body irradiated mice

    International Nuclear Information System (INIS)

    Ina, Yasuhiro; Sakai, Kazuo

    2003-01-01

    The effects of low-dose rate whole body irradiation on biodefense and immunological systems were investigated using female C57BL/6 (B6) mice. These B6 mice were exposed continuously to γ-rays from a 137 Cs source in the long-term low-dose rate irradiation facility at CRIEPI for 0 - 12 weeks at a dose rate of 0.95 mGy/hr. In the bone marrow, thymus, spleen, lymph nodes, and peripheral blood of the irradiated mice, changes in cell populations and cell surface molecules were examined. The cell surface functional molecules (CD3, CD4, CD8, CD19, CD45R/B220, ICAM-1, Fas, NK-1.1, CXCR4, and CCR5), and activation molecules (THAM, CD28, CD40, CD44H, CD70, B7-1, B7-2, OX-40 antigen, CTLA-4, CD30 ligand, and CD40 ligand) were analyzed by flow cytometry. The percentage of CD4 + T cells and cell surface CD8 molecule expressions on the CD8 + T cells increased significantly to 120-130% after 3 weeks of the irradiation, compared to non-irradiated control mice. On the other hand, the percentage of CD45R/B220 + CD40 + B cells, which is one of the immunological markers of inflammation, infection, tumor, and autoimmune disease, decreased significantly to 80-90% between the 3rd to 5th week of irradiation. There was no significant difference in other cell population rates and cell surface molecule expression. Furthermore, abnormal T cells bearing mutated T cell receptors induced by high-dose rate irradiation were not observed throughout this study. These results suggest that low-dose rate irradiation activates the immunological status of the whole body. (author)

  20. Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

    International Nuclear Information System (INIS)

    Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

    2010-01-01

    A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

  1. TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

    Science.gov (United States)

    Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

    2012-02-01

    TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

  2. Heritability and genetic basis of protein level variation in an outbred population.

    Science.gov (United States)

    Parts, Leopold; Liu, Yi-Chun; Tekkedil, Manu M; Steinmetz, Lars M; Caudy, Amy A; Fraser, Andrew G; Boone, Charles; Andrews, Brenda J; Rosebrock, Adam P

    2014-08-01

    The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population's average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. © 2014 Parts et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    Science.gov (United States)

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  4. Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells

    Science.gov (United States)

    Hao, Wen-Rui; Sung, Li-Chin; Chen, Chun-Chao; Chen, Jin-Jer

    2018-01-01

    Moderate coffee consumption is inversely associated with cardiovascular disease mortality; however, mechanisms underlying this causal effect remain unclear. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. This study investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion in vascular endothelial cells. Cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinases (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase and p38 MAPK. By contrast, cafestol upregulated cyclic-strain-induced HO-1 and Sirt1 expression. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly attenuated cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. This is the first study to report that cafestol inhibited cyclic-strain-induced inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol. PMID:29854096

  5. Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Di [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Yuan, Yunsheng [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai (China); Chen, Li [Pharmacy College, Fujian University of Traditional Chinese Medicine, Fuzhou (China); Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Liu, Xin; Belani, Chandra [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Cheng, Hua, E-mail: hcheng@ihv.umaryland.edu [Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Department Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States)

    2015-08-14

    Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and IκB kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells. - Highlights: • Niclosamide is a promising therapeutic candidate for adult T cell leukemia. • Niclosamide employs a novel mechanism through proteasomal degradation of Tax. • Niclosamide downregulates certain cellular pro-survival molecules.

  6. A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

    Science.gov (United States)

    2015-01-01

    Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

  7. Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

    International Nuclear Information System (INIS)

    Liepinsh, Edvards; Sodano, Patrick; Tassin, Severine; Marion, Didier; Vovelle, Francoise; Otting, Gottfried

    1999-01-01

    Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane

  8. Sources of dietary protein in relation to blood pressure in a general Dutch population.

    Directory of Open Access Journals (Sweden)

    Wieke Altorf-van der Kuil

    Full Text Available BACKGROUND: Little is known about the relation of different dietary protein types with blood pressure (BP. We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20-65 y and not using antihypertensive medication. DESIGN: Mean BP levels were calculated in quintiles of energy-adjusted protein with adjustment for age, sex, BMI, education, smoking, and intake of energy, alcohol, and other nutrients including protein from other sources. In addition, mean BP difference after substitution of 3 en% carbohydrates or MUFA with protein was calculated. RESULTS: Total protein and animal protein were not associated with BP (p(trend = 0.62 and 0.71 respectively, both at the expense of carbohydrates and MUFA. Systolic BP was 1.8 mmHg lower (p(trend36 g/d than in the lowest (<27 g/d quintile of plant protein. This inverse association was present both at the expense of carbohydrates and MUFA and more pronounced in individuals with untreated hypertension (-3.6 mmHg than in those with normal (+0.1 mmHg or prehypertensive BP (-0.3 mmHg; p(interaction<0.01. Meat and grain protein were not related to BP. Dairy protein was directly associated with systolic BP (+1.6 mmHg, p(trend<0.01, but not with diastolic BP (p(trend = 0.24. CONCLUSIONS: Total protein and animal protein were not associated with BP in this general untreated Dutch population. Plant protein may be beneficial to BP, especially in people with elevated BP. However, because high intake of plant protein may be a marker of a healthy diet and lifestyle in general, confirmation from randomized controlled trials is warranted.

  9. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Monti, Paola; Fronza, Gilberto [Mutagenesis Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa (Italy); Pereira, Clara [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Saraiva, Lucília, E-mail: lucilia.saraiva@ff.up.pt [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal)

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  10. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    International Nuclear Information System (INIS)

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73

  11. Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

    Science.gov (United States)

    Cui, Yunxi; Koirala, Deepak; Kang, HyunJin; Dhakal, Soma; Yangyuoru, Philip; Hurley, Laurence H; Mao, Hanbin

    2014-05-01

    Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

  12. Assessing Genetic Diversity Based on Gliadin Proteins in Aegilops cylindrica Populations from Northwest of Iran

    Directory of Open Access Journals (Sweden)

    Toraj KHABIRI

    2013-02-01

    Full Text Available Wild wheat progenitors served as a valuable gene pool in breeding perspectives. In this respect, gliadins could be an important tool in assessing genetic variability as protein markers. Thus, genetic diversity of gliadin protein patterns in seventeen populations of Aegilops cylindrica collected from northwest of Iran were investigated using acid polyacrylamide gel electrophoresis. Results showed that the highest number of bands in the electrophoregrams were related to the ω type of geliadins. Conversely, the lowest number of bands were pertained to the β type of gliadins. Genetic diversity between populations was greater than within population variation. Assessment of total variation for the three gliadin types indicated that the highest total variation was related to β type while, the lowest one was belonged to ω type. Cluster analysis using complete linkage method divided populations into two separated groups in which genetic diversity does not follow from geographical distribution.

  13. Discovery of an Unexplored Protein Structural Scaffold of Serine Protease from Big Blue Octopus (Octopus cyanea): A New Prospective Lead Molecule.

    Science.gov (United States)

    Panda, Subhamay; Kumari, Leena

    2017-01-01

    Serine proteases are a group of enzymes that hydrolyses the peptide bonds in proteins. In mammals, these enzymes help in the regulation of several major physiological functions such as digestion, blood clotting, responses of immune system, reproductive functions and the complement system. Serine proteases obtained from the venom of Octopodidae family is a relatively unexplored area of research. In the present work, we tried to effectively utilize comparative composite molecular modeling technique. Our key aim was to propose the first molecular model structure of unexplored serine protease 5 derived from big blue octopus. The other objective of this study was to analyze the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the aid of different bioinformatic tools. In the present study, molecular model has been generated with the help of I-TASSER suite. Afterwards the refined structural model was validated with standard methods. For functional annotation of protein molecule we used Protein Information Resource (PIR) database. Serine protease 5 of big blue octopus was analyzed with different bioinformatical algorithms for the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. The molecular model data in cooperation to other pertinent post model analysis data put forward molecular insight to proteolytic activity of serine protease 5, which helps in the clear understanding of procoagulant and anticoagulant characteristics of this natural lead molecule. Our approach was to investigate the octopus

  14. Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

    Science.gov (United States)

    Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

    2015-10-22

    One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

  15. Natural compounds as a source of protein tyrosine phosphatase inhibitors : Application to the rational design of small-molecule derivatives

    NARCIS (Netherlands)

    Ferreira, Carmen V.; Justo, Giselle Z.; Souza, Ana C. S.; Queiroz, Karla C. S.; Zambuzzi, William F.; Aoyama, Hiroshi; Peppelenbosch, Maikel P.

    2006-01-01

    Reversible phosphorylation of tyrosine residues is a key regulatory mechanism for numerous cellular events. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) have a pivotal role in regulating both normal cell physiology and pathophysiology. Accordingly, deregulated activity of both

  16. Patterns of Protein Food Intake Are Associated with Nutrient Adequacy in the General French Adult Population.

    Science.gov (United States)

    Gavelle, Erwan de; Huneau, Jean-François; Mariotti, François

    2018-02-17

    Protein food intake appears to partially structure dietary patterns, as most current emergent diets (e.g., vegetarian and flexitarian) can be described according to their levels of specific protein sources. However, few data are available on dietary protein patterns in the general population and their association with nutrient adequacy. Based on protein food intake data concerning 1678 adults from a representative French national dietary survey, and non-negative-matrix factorization followed by cluster analysis, we were able to identify distinctive dietary protein patterns and compare their nutrient adequacy (using PANDiet probabilistic scoring). The findings revealed eight patterns that clearly discriminate protein intakes and were characterized by the intakes of one or more specific protein foods: 'Processed meat', 'Poultry', 'Pork', 'Traditional', 'Milk', 'Take-away', 'Beef' and 'Fish'. 'Fish eaters' and 'Milk drinkers' had the highest overall nutrient adequacy, whereas that of 'Pork' and 'Take-away eaters' was the lowest. Nutrient adequacy could often be accounted for by the characteristics of the food contributing to protein intake: 'Meat eaters' had high probability of adequacy for iron and zinc, for example. We concluded that protein patterns constitute strong elements in the background structure of the dietary intake and are associated with the nutrient profile that they convey.

  17. Cell-killing efficiency and number of platinum atoms binding to DNA, RNA and protein molecules of HeLa cells treated with combinations of hyperthermia and carboplatin

    International Nuclear Information System (INIS)

    Akaboshi, M.; Kawai, K.; Tanaka, Y.; Takada, J.; Sumino, T.

    1999-01-01

    The effect of hyperthermia on the cell killing efficiency of Pt atoms binding to DNA, RNA and protein molecules of HeLa cells treated with cis-diamine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was examined. HeLa S-3 cells were treated with 195m Pt-radiolabeled CBDCA for 60 minutes at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentration (D 0 ) of carboplatin for a 60 min-treatment at 0, 25, 37, 40, 42 and 44 deg C was 671.2, 201.5, 67.3, 33.4, 20.2 and 15.6 μM, respectively. By using identically treated cells, the number of Pt-atoms combined with DNA, RNA and protein molecules were determined in the subcellular fractions. Thus, the D 0 's given as the drug concentrations were replaced with the number of Pt-atoms combined in each fraction. Then, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt-atoms combined and was calculated for each molecule. The efficiency for DNA molecules was 0.699, 1.42, 2.65, 4.84, 7.74 and 8.28x10 4 nucleotides, respectively, for the conditions described above. From 0 to 44 deg C, the cell-killing efficiency of Pt atoms increased by a factor of 11.9. (author)

  18. Chemical biology based on target-selective degradation of proteins and carbohydrates using light-activatable organic molecules.

    Science.gov (United States)

    Toshima, Kazunobu

    2013-05-01

    Proteins and carbohydrates play crucial roles in a wide range of biological processes, including serious diseases. The development of novel and innovative methods for selective control of specific proteins and carbohydrates functions has attracted much attention in the field of chemical biology. In this account article, the development of novel chemical tools, which can degrade target proteins and carbohydrates by irradiation with a specific wavelength of light under mild conditions without any additives, is introduced. This novel class of photochemical agents promise bright prospects for finding not only molecular-targeted bioprobes for understanding of the structure-activity relationships of proteins and carbohydrates but also novel therapeutic drugs targeting proteins and carbohydrates.

  19. Interaction Research on the Antiviral Molecule Dufulin Targeting on Southern Rice Black Streaked Dwarf Virus P9-1 Nonstructural Protein

    Directory of Open Access Journals (Sweden)

    Zhenchao Wang

    2015-03-01

    Full Text Available ern rice black streaked dwarf virus (SRBSDV causes severe harm to rice production. Unfortunately, studies on effective antiviral drugs against SRBSDV and interaction mechanism of antiviral molecule targeting on SRBSDV have not been reported. This study found dufulin (DFL, an ideal anti-SRBSDV molecule, and investigated the interactions of DFL targeting on the nonstructural protein P9-1. The biological sequence information and bonding characterization of DFL to four kinds of P9-1 protein were described with fluorescence titration (FT and microscale thermophoresis (MST assays. The sequence analysis indicated that P9-1 had highly-conserved C- and N-terminal amino acid residues and a hypervariable region that differed from 131 aa to 160 aa. Consequently, wild-type (WT-His-P9-1, 23 C-terminal residues truncated (TR-ΔC23-His-P9-1, 6 N-terminal residues truncated (TR-ΔN6-His-P9-1, and Ser138 site-directed (MU-138-His-P9-1 mutant proteins were expressed. The FT and MST assay results indicated that DFL bounded to WT-His-P9-1 with micromole affinity and the 23 C-terminal amino acids were the potential targeting site. This system, which combines a complete sequence analysis, mutant protein expression, and binding action evaluating system, could further advance the understanding of the interaction abilities between antiviral drugs and their targets.

  20. Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein

    International Nuclear Information System (INIS)

    Petty, Tom J.; Nishimura, Taisuke; Emamzadah, Soheila; Gabus, Caroline; Paszkowski, Jerzy; Halazonetis, Thanos D.; Thore, Stéphane

    2010-01-01

    In order to investigate its function in transcriptional gene silencing, the highly conserved motif 2 from A. thaliana Morpheus’ molecule 1 protein was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 3.2 Å. Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing

  1. Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

    OpenAIRE

    Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

    2010-01-01

    We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is...

  2. Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Skovgaard, Kerstin; Heegaard, Peter M. H.

    2011-01-01

    Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated...... the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam3Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN...

  3. Selective Advantage of Recombination in Evolving Protein Populations:. a Lattice Model Study

    Science.gov (United States)

    Williams, Paul D.; Pollock, David D.; Goldstein, Richard A.

    Recent research has attempted to clarify the contributions of several mutational processes, such as substitutions or homologous recombination. Simplistic, tractable protein models, which determine the compact native structure phenotype from the sequence genotype, are well-suited to such studies. In this paper, we use a lattice-protein model to examine the effects of point mutation and homologous recombination on evolving populations of proteins. We find that while the majority of mutation and recombination events are neutral or deleterious, recombination is far more likely to be beneficial. This results in a faster increase in fitness during evolution, although the final fitness level is not significantly changed. This transient advantage provides an evolutionary advantage to subpopulations that undergo recombination, allowing fixation of recombination to occur in the population.

  4. A mechanistic approach to link biological effects of radioactive substances from molecules to populations in wildlife species - A mechanistic approach to link biological effects of radionuclides from molecules to populations in wildlife species

    Energy Technology Data Exchange (ETDEWEB)

    Alonzo, Frederic; Parisot, Florian; Plaire, Delphine; Adam-Guillermin, Christelle; Garnier- Laplace, Jacqueline [Institut de Radioprotection et de Surete Nucleaire (IRSN), PRP-ENV, SERIS, LECO, Cadarache, Saint-Paul- Lez-Durance, 13115 (France)

    2014-07-01

    Understanding how toxic contaminants affect wildlife species at various levels of biological organisation (sub-cellular, histological, physiological, organism, population levels) is a major research goal in both ecotoxicology and radioecology. A mechanistic understanding of the links between the different observed perturbations is necessary to predict consequences for survival, growth and reproduction which are critical for population dynamics. However, time scales at which such links are established in the laboratory are rarely relevant for natural populations. With a small size and short life cycle, the cladoceran micro-crustacean Daphnia magna is a particularly suitable biological model for studying effects of radioactive contaminants over several generations. Multi-generational exposures are much more representative of the environmental context of field populations for which contaminations can last for durations which largely exceed individual longevity and involve exposure of many successive generations. Over the last decade, multi-generational investigations of toxic effects were conducted under controlled conditions in D. magna exposed to various radionuclides including depleted uranium, americium-241 and cesium-137, representing respectively a dominantly chemo-toxic metal, an alpha internal contamination and a gamma external radiation. Results showed in all cases that toxic effects on physiology and life history (survival, body size, fecundity) increased in severity across generations. These observations demonstrated that measured effects in one generation might not be representative of toxicity in the following offspring generations, and ultimately of the population response. Reduction in somatic growth and reproduction induced by uranium were analysed using the mechanistic modelling approach known as DEBtox (model of dynamic energy budget applied to toxicology). Modelling results suggested that uranium primarily affects assimilation. This metabolic mode

  5. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  6. Spectroscopic diagnostics of the vibrational population in the ground state of H2 and D2 molecules

    International Nuclear Information System (INIS)

    Fantz, U.; Heger, B.

    1998-01-01

    A diagnostic method has been evaluated for measuring the relative vibrational ground-state population of molecular hydrogen and deuterium. It is based on the analysis of the diagonal Fulcher bands · 3 Π u →a 3 Σ g + ) and the Franck-Condon principle of excitation. The validity of the underlying assumptions was verified by experiments in microwave discharges and the method is recommended for application in divertor plasmas in controlled fusion experiments. By attributing a vibrational temperature T vib to the ground-state electronic level (X 1 Σ g + ) and assuming population via the Franck-Condon principle, the upper Fulcher state vibrational distribution can be derived theoretically with T vib as parameter. Comparison with experimentally derived upper-state population gives the corresponding T vib of the ground state. The Franck-Condon factors for the · 3 Π 1 Σ g + and · 3 Π u →a 3 Σ g + transitions have been calculated for both hydrogen and deuterium from molecular constants using the FCFRKR code. The method has been applied to low pressure H 2 /He and D 2 /He microwave plasmas, showing good agreement of experimentally and theoretically derived upper Fulcher state vibrational distributions. The vibrational temperatures range from 3200 K to 6800 K for H 2 and 2600 K to 4000 K for D 2 · depending on molecular density, pressure and electron temperature, but indicating nearly the same vibrational population for H 2 and D 2 for comparable plasma conditions. (author)

  7. Changes of Serum Intercellular Adhesion Molecule – 1, Vascular Adhesion Molecule-1 and C – Reactive Protein in Middle-Aged Men with Heart Failure after Eight Weeks of Aerobic Exercise

    Directory of Open Access Journals (Sweden)

    Hoda Haghir

    2017-03-01

    Full Text Available Introduction: The evidence has shown that expansion of cardiovascular disease has inflammation base, and general inflammation (systemic plays a pivotal role in the development of atherosclerosis. The purpose of this research was evaluation of changes in intercellular adhesion molecule – 1, vascular adhesion molecule-1 and C – reactive protein in middle-aged men with heart failure after eight weeks of aerobic exercise. Methods: Twenty four middle-aged men with heart failure were selected as volunteers, and were divided into two groups; the aerobic training and the control groups. Aerobic training program was eight weeks, three times per week with the intensity of 40%-70% maximum heart rate. Fasting blood samples were taken from all subjects before and after eight weeks of aerobic exercise. . Data were analyzed by paired sample t-test and independent sample t-test at a significance levels of P<0.05. Results: In the aerobic training group, comparison within groups showed, serum levels of ICAM-1, VCAM-1 and CRP (respectively P=0.001, P=0.001 and P=0.001 were significantly reduced. There was a significant reduction in comparison between groups only for VCAM-1 (P=0.001 and CRP (P=0.002. Conclusion: Aerobic exercise with reducing levels of inflammatory markers ICAM-1 and CRP may play an important role in the prevention and control of cardiovascular diseases in middle-aged men with heart failure.

  8. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

    Directory of Open Access Journals (Sweden)

    Loren H. Rieseberg

    2012-10-01

    Full Text Available Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq to refine our understanding of the population genomics of wild species of sunflowers (Helianthus spp. and the factors that affect rates of protein evolution. We aligned 35 GB of transcriptome sequencing data and identified 433,257 polymorphic sites (SNPs in a reference transcriptome comprising 16,312 genes. Using SNP markers, we identified strong population clustering largely corresponding to the three species analyzed here (Helianthus annuus, H. petiolaris, H. debilis, with one distinct early generation hybrid. Then, we calculated the proportions of adaptive substitution fixed by selection (alpha and identified gene ontology categories with elevated values of alpha. The “response to biotic stimulus” category had the highest mean alpha across the three interspecific comparisons, implying that natural selection imposed by other organisms plays an important role in driving protein evolution in wild sunflowers. Finally, we examined the relationship between protein evolution (dN/dS ratio and several genomic factors predicted to co-vary with protein evolution (gene expression level, divergence and specificity, genetic divergence [FST], and nucleotide diversity pi. We find that variation in rates of protein divergence was correlated with gene expression level and specificity, consistent with results from a broad range of taxa and timescales. This would in turn imply that these factors govern protein evolution both at a microevolutionary and macroevolutionary timescale. Our results contribute to a general understanding of the

  9. Chemical effects of ionizing radiation on the individual amino acids within intact and pure protein molecules. Final report

    International Nuclear Information System (INIS)

    Freidberg, F.

    1977-01-01

    Progress is reported on the following research projects: gamma radiation induced chemical and molecular weight changes in proteins; the free radical pattern for the irradiated protein; similarities in the mechanism of action of ionizing and of uv radiation; and spin trapping in the study of gamma radiolysis

  10. Genetic Variation and Population Structure in Jamunapari Goats Using Microsatellites, Mitochondrial DNA, and Milk Protein Genes

    Science.gov (United States)

    Rout, P. K.; Thangraj, K.; Mandal, A.; Roy, R.

    2012-01-01

    Jamunapari, a dairy goat breed of India, has been gradually declining in numbers in its home tract over the years. We have analysed genetic variation and population history in Jamunapari goats based on 17 microsatellite loci, 2 milk protein loci, mitochondrial hypervariable region I (HVRI) sequencing, and three Y-chromosomal gene sequencing. We used the mitochondrial DNA (mtDNA) mismatch distribution, microsatellite data, and bottleneck tests to infer the population history and demography. The mean number of alleles per locus was 9.0 indicating that the allelic variation was high in all the loci and the mean heterozygosity was 0.769 at nuclear loci. Although the population size is smaller than 8,000 individuals, the amount of variability both in terms of allelic richness and gene diversity was high in all the microsatellite loci except ILST 005. The gene diversity and effective number of alleles at milk protein loci were higher than the 10 other Indian goat breeds that they were compared to. Mismatch analysis was carried out and the analysis revealed that the population curve was unimodal indicating the expansion of population. The genetic diversity of Y-chromosome genes was low in the present study. The observed mean M ratio in the population was above the critical significance value (Mc) and close to one indicating that it has maintained a slowly changing population size. The mode-shift test did not detect any distortion of allele frequency and the heterozygosity excess method showed that there was no significant departure from mutation-drift equilibrium detected in the population. However, the effects of genetic bottlenecks were observed in some loci due to decreased heterozygosity and lower level of M ratio. There were two observed genetic subdivisions in the population supporting the observations of farmers in different areas. This base line information on genetic diversity, bottleneck analysis, and mismatch analysis was obtained to assist the conservation

  11. A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface

    International Nuclear Information System (INIS)

    Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

    2013-01-01

    The triclinic structure of the HIV-1 capsid protein contains four molecules in the asymmetric unit that form a novel packing interface that could conceivably resemble an intermediate structure that is involved in the early steps of HIV-1 assembly. The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs

  12. Small Molecules from Nature Targeting G-Protein Coupled Cannabinoid Receptors: Potential Leads for Drug Discovery and Development

    Directory of Open Access Journals (Sweden)

    Charu Sharma

    2015-01-01

    Full Text Available The cannabinoid molecules are derived from Cannabis sativa plant which acts on the cannabinoid receptors types 1 and 2 (CB1 and CB2 which have been explored as potential therapeutic targets for drug discovery and development. Currently, there are numerous cannabinoid based synthetic drugs used in clinical practice like the popular ones such as nabilone, dronabinol, and Δ9-tetrahydrocannabinol mediates its action through CB1/CB2 receptors. However, these synthetic based Cannabis derived compounds are known to exert adverse psychiatric effect and have also been exploited for drug abuse. This encourages us to find out an alternative and safe drug with the least psychiatric adverse effects. In recent years, many phytocannabinoids have been isolated from plants other than Cannabis. Several studies have shown that these phytocannabinoids show affinity, potency, selectivity, and efficacy towards cannabinoid receptors and inhibit endocannabinoid metabolizing enzymes, thus reducing hyperactivity of endocannabinoid systems. Also, these naturally derived molecules possess the least adverse effects opposed to the synthetically derived cannabinoids. Therefore, the plant based cannabinoid molecules proved to be promising and emerging therapeutic alternative. The present review provides an overview of therapeutic potential of ligands and plants modulating cannabinoid receptors that may be of interest to pharmaceutical industry in search of new and safer drug discovery and development for future therapeutics.

  13. Expanding the Cancer Arsenal with Targeted Therapies: Disarmament of the Antiapoptotic Bcl-2 Proteins by Small Molecules.

    Science.gov (United States)

    Yap, Jeremy L; Chen, Lijia; Lanning, Maryanna E; Fletcher, Steven

    2017-02-09

    A hallmark of cancer is the evasion of apoptosis, which is often associated with the upregulation of the antiapoptotic members of the Bcl-2 family of proteins. The prosurvival function of the antiapoptotic Bcl-2 proteins is manifested by capturing and neutralizing the proapoptotic Bcl-2 proteins via their BH3 death domains. Accordingly, strategies to antagonize the antiapoptotic Bcl-2 proteins have largely focused on the development of low-molecular-weight, synthetic BH3 mimetics ("magic bullets") to disrupt the protein-protein interactions between anti- and proapoptotic Bcl-2 proteins. In this way, apoptosis has been reactivated in malignant cells. Moreover, several such Bcl-2 family inhibitors are presently being evaluated for a range of cancers in clinical trials and show great promise as new additions to the cancer armamentarium. Indeed, the selective Bcl-2 inhibitor venetoclax (Venclexta) recently received FDA approval for the treatment of a specific subset of patients with chronic lymphocytic leukemia. This review focuses on the major developments in the field of Bcl-2 inhibitors over the past decade, with particular emphasis on binding modes and, thus, the origins of selectivity for specific Bcl-2 family members.

  14. An Augmented Pocketome: Detection and Analysis of Small-Molecule Binding Pockets in Proteins of Known 3D Structure.

    Science.gov (United States)

    Bhagavat, Raghu; Sankar, Santhosh; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2018-03-06

    Protein-ligand interactions form the basis of most cellular events. Identifying ligand binding pockets in proteins will greatly facilitate rationalizing and predicting protein function. Ligand binding sites are unknown for many proteins of known three-dimensional (3D) structure, creating a gap in our understanding of protein structure-function relationships. To bridge this gap, we detect pockets in proteins of known 3D structures, using computational techniques. This augmented pocketome (PocketDB) consists of 249,096 pockets, which is about seven times larger than what is currently known. We deduce possible ligand associations for about 46% of the newly identified pockets. The augmented pocketome, when subjected to clustering based on similarities among pockets, yielded 2,161 site types, which are associated with 1,037 ligand types, together providing fold-site-type-ligand-type associations. The PocketDB resource facilitates a structure-based function annotation, delineation of the structural basis of ligand recognition, and provides functional clues for domains of unknown functions, allosteric proteins, and druggable pockets. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

    Science.gov (United States)

    Risco, Cristina; Sanmartín-Conesa, Eva; Tzeng, Wen-Pin; Frey, Teryl K.; Seybold, Volker; de Groot, Raoul J.

    2012-01-01

    Summary More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell’s interior are lagging behind. We describe a novel approach, metal-tagging TEM (METTEM) that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before. PMID:22579245

  16. Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

    Czech Academy of Sciences Publication Activity Database

    Harris, N.; Kogan, F. Y.; Ilková, G.; Juhás, Štefan; Lahmy, O.; Gregor, Y. I.; Koppel, J.; Zhuk, R.; Gregor, P.

    2014-01-01

    Roč. 1840, č. 1 (2014), s. 245-254 ISSN 0304-4165 Institutional support: RVO:67985904 Keywords : heparan sulfate * heparin binding protein * glycosaminoglycan Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.381, year: 2014

  17. Regulation of membrane protein function by lipid bilayer elasticity—a single molecule technology to measure the bilayer properties experienced by an embedded protein

    DEFF Research Database (Denmark)

    Lundbæk, Jens August

    2008-01-01

    , regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes......-dependent sodium channels, N-type calcium channels and GABAA receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic...... properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established....

  18. Regulation of membrane protein function by lipid bilayer elasticity-a single molecule technology to measure the bilayer properties experienced by an embedded protein

    International Nuclear Information System (INIS)

    Lundbaek, Jens August

    2006-01-01

    Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes, in the general regulation of membrane protein function, is unclear. This is to a large extent due to lack of a generally accepted framework in which to understand the many observations. The present review summarizes studies which have demonstrated that the hydrophobic interactions between a membrane protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this 'hydrophobic coupling mechanism' has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using voltage-dependent sodium channels, N-type calcium channels and GABA A receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established

  19. Virtual screening of natural inhibitors to the predicted HBx protein structure of Hepatitis B Virus using molecular docking for identification of potential lead molecules for liver cancer

    Science.gov (United States)

    Pathak, Rajesh Kumar; Baunthiyal, Mamta; Taj, Gohar; Kumar, Anil

    2014-01-01

    The HBx protein in Hepatitis B Virus (HBV) is a potential target for anti-liver cancer molecules. Therefore, it is of interest to screen known natural compounds against the HBx protein using molecular docking. However, the structure of HBx is not yet known. Therefore, the predicted structure of HBx using threading in LOMET was used for docking against plant derived natural compounds (curcumin, oleanolic acid, resveratrol, bilobetin, luteoline, ellagic acid, betulinic acid and rutin) by Molegro Virtual Docker. The screening identified rutin with binding energy of -161.65 Kcal/mol. Thus, twenty derivatives of rutin were further designed and screened against HBx. These in silico experiments identified compounds rutin01 (-163.16 Kcal/mol) and rutin08 (- 165.76 Kcal/mol) for further consideration and downstream validation. PMID:25187683

  20. Identification of the Ulex europaeus agglutinin-I-binding protein as a unique glycoform of the neural cell adhesion molecule in the olfactory sensory axons of adults rats.

    Science.gov (United States)

    Pestean, A; Krizbai, I; Böttcher, H; Párducz, A; Joó, F; Wolff, J R

    1995-08-04

    Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.

  1. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    Science.gov (United States)

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  2. Human skeletal muscle type 1 fibre distribution and response of stress-sensing proteins along the titin molecule after submaximal exhaustive exercise.

    Science.gov (United States)

    Koskinen, Satu O A; Kyröläinen, Heikki; Flink, Riina; Selänne, Harri P; Gagnon, Sheila S; Ahtiainen, Juha P; Nindl, Bradley C; Lehti, Maarit

    2017-11-01

    Early responses of stress-sensing proteins, muscle LIM protein (MLP), ankyrin repeat proteins (Ankrd1/CARP and Ankrd2/Arpp) and muscle-specific RING finger proteins (MuRF1 and MuRF2), along the titin molecule were investigated in the present experiment after submaximal exhaustive exercise. Ten healthy men performed continuous drop jumping unilaterally on a sledge apparatus with a submaximal height until complete exhaustion. Five stress-sensing proteins were analysed by mRNA measurements from biopsies obtained immediately and 3 h after the exercise from exercised vastus lateralis muscle while control biopsies were obtained from non-exercised legs before the exercise. Decreased maximal jump height and increased serum creatine kinase activities as indirect markers for muscle damage and HSP27 immunostainings on muscle biopsies as a direct marker for muscle damage indicated that the current exercised protocol caused muscle damage. mRNA levels for four (MLP, Ankrd1/CARP, MuRF1 and MuRF2) out of the five studied stress sensors significantly (p exercise. The magnitude of MLP and Ankrd2 responses was related to the proportion of type 1 myofibres. Our data showed that the submaximal exhaustive exercise with subject's own physical fitness level activates titin-based stretch-sensing proteins. These results suggest that both degenerative and regenerative pathways are activated in very early phase after the exercise or probably already during the exercise. Activation of these proteins represents an initial step forward adaptive remodelling of the exercised muscle and may also be involved in the initiation of myofibre repair.

  3. [Dynamics of Irreversible Evaporation of a Water-Protein Droplet and a Problem of Structural and Dynamical Experiments with Single Molecules].

    Science.gov (United States)

    Shaitan, K V; Armeev, G A; Shaytan, A K

    2016-01-01

    We discuss the effect of isothermal and adiabatic evaporation of water on the state of a water-protein droplet. The discussed problem is of current importance due to development of techniques to perform single molecule experiments using free electron lasers. In such structure-dynamic experiments the delivery of a sample into the X-ray beam is performed using the microdroplet injector. The time between the injection and delivery is in the order of microseconds. In this paper we developed a specialized variant of all-atom molecular dynamics simulations for the study of irreversible isothermal evaporation of the droplet. Using in silico experiments we determined the parameters of isothermal evaporation of the water-protein droplet with the sodium and chloride ions in the concentration range of 0.3 M at different temperatures. The energy of irreversible evaporation determined from in silico experiments at the initial stages of evaporation virtually coincides with the specific heat of evaporation for water. For the kinetics of irreversible adiabatic evaporation an exact analytical solution was obtained in the limit of high thermal conductivity of the droplet (or up to the droplet size of -100 Å). This analytical solution incorporates parameters that are determined using in silico. experiments on isothermal droplet evaporation. We show that the kinetics of adiabatic evaporation and cooling of the droplet scales with the droplet size. Our estimates of the water-protemi droplet. freezing rate in the adiabatic regime in a vacuum chamber show that additional techniques for stabilizing the temperature inside the droplet should be used in order to study the conformational transitions of the protein in single molecules. Isothermal and quasi-isothermal conditions are most suitable for studying the conformational transitions upon object functioning. However, in this case it is necessary to take into account the effects of dehydration and rapid increase of ionic strength in an

  4. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  5. Infrared laser induced population transfer and parity selection in (14)NH3: A proof of principle experiment towards detecting parity violation in chiral molecules.

    Science.gov (United States)

    Dietiker, P; Miloglyadov, E; Quack, M; Schneider, A; Seyfang, G

    2015-12-28

    We have set up an experiment for the efficient population transfer by a sequential two photon-absorption and stimulated emission-process in a molecular beam to prepare quantum states of well defined parity and their subsequent sensitive detection. This provides a proof of principle for an experiment which would allow for parity selection and measurement of the time evolution of parity in chiral molecules, resulting in a measurement of the parity violating energy difference ΔpvE between enantiomers of chiral molecules. Here, we present first results on a simple achiral molecule demonstrating efficient population transfer (about 80% on the average for each step) and unperturbed persistence of a selected excited parity level over flight times of about 1.3 ms in the beam. In agreement with model calculations with and without including nuclear hyperfine structure, efficient population transfer can be achieved by a rather simple implementation of the rapid adiabatic passage method of Reuss and coworkers and considering also the stimulated Raman adiabatic passage technique of Bergmann and coworkers as an alternative. The preparation step uses two powerful single mode continuous wave optical parametric oscillators of high frequency stability and accuracy. The detection uses a sensitive resonantly enhanced multiphoton ionization method after free flight lengths of up to 0.8 m in the molecular beam. Using this technique, we were able to also resolve the nuclear hyperfine structure in the rovibrational levels of the ν1 and ν3 fundamentals as well as the 2ν4 overtone of (14)NH3, for which no previous data with hyperfine resolution were available. We present our new results on the quadrupole coupling constants for the ν1, ν3, and 2ν4 levels in the context of previously known data for ν2 and its overtone, as well as ν4, and the ground state. Thus, now, (14)N quadrupole coupling constants for all fundamentals and some overtones of (14)NH3 are known and can be used for

  6. Infrared laser induced population transfer and parity selection in {sup 14}NH{sub 3}: A proof of principle experiment towards detecting parity violation in chiral molecules

    Energy Technology Data Exchange (ETDEWEB)

    Dietiker, P.; Miloglyadov, E.; Quack, M., E-mail: Martin@Quack.ch; Schneider, A.; Seyfang, G. [Physical Chemistry, ETH Zürich, CH-8093 Zürich (Switzerland)

    2015-12-28

    We have set up an experiment for the efficient population transfer by a sequential two photon—absorption and stimulated emission—process in a molecular beam to prepare quantum states of well defined parity and their subsequent sensitive detection. This provides a proof of principle for an experiment which would allow for parity selection and measurement of the time evolution of parity in chiral molecules, resulting in a measurement of the parity violating energy difference Δ{sub pv}E between enantiomers of chiral molecules. Here, we present first results on a simple achiral molecule demonstrating efficient population transfer (about 80% on the average for each step) and unperturbed persistence of a selected excited parity level over flight times of about 1.3 ms in the beam. In agreement with model calculations with and without including nuclear hyperfine structure, efficient population transfer can be achieved by a rather simple implementation of the rapid adiabatic passage method of Reuss and coworkers and considering also the stimulated Raman adiabatic passage technique of Bergmann and coworkers as an alternative. The preparation step uses two powerful single mode continuous wave optical parametric oscillators of high frequency stability and accuracy. The detection uses a sensitive resonantly enhanced multiphoton ionization method after free flight lengths of up to 0.8 m in the molecular beam. Using this technique, we were able to also resolve the nuclear hyperfine structure in the rovibrational levels of the ν{sub 1} and ν{sub 3} fundamentals as well as the 2ν{sub 4} overtone of {sup 14}NH{sub 3}, for which no previous data with hyperfine resolution were available. We present our new results on the quadrupole coupling constants for the ν{sub 1}, ν{sub 3}, and 2ν{sub 4} levels in the context of previously known data for ν{sub 2} and its overtone, as well as ν{sub 4}, and the ground state. Thus, now, {sup 14}N quadrupole coupling constants for all

  7. Low-temperature FTIR spectroscopy provides evidence for protein-bound water molecules in eubacterial light-driven ion pumps.

    Science.gov (United States)

    Nomura, Yurika; Ito, Shota; Teranishi, Miwako; Ono, Hikaru; Inoue, Keiichi; Kandori, Hideki

    2018-01-31

    Light-driven H + , Na + and Cl - pumps have been found in eubacteria, which convert light energy into a transmembrane electrochemical potential. A recent mutation study revealed asymmetric functional conversion between the two pumps, where successful functional conversions are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Although this fact suggests that the essential structural mechanism of an ancestral function is retained even after gaining a new function, questions regarding the essential structural mechanism remain unanswered. Light-induced difference FTIR spectroscopy was used to monitor the presence of strongly hydrogen-bonded water molecules for all eubacterial H + , Na + and Cl - pumps, including a functionally converted mutant. This fact suggests that the strongly hydrogen-bonded water molecules are maintained for these new functions during evolution, which could be the reason for successful functional conversion from Na + to H + , and from Cl - to H + pumps. This also explains the successful conversion of the Cl - to the H + pump only for eubacteria, but not for archaea. It is concluded that water-containing hydrogen-bonding networks constitute one of the essential structural mechanisms in eubacterial light-driven ion pumps.

  8. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

  9. Health effects of protein intake in healthy elderly populations: a systematic literature review

    Directory of Open Access Journals (Sweden)

    Agnes N. Pedersen

    2014-02-01

    Full Text Available The purpose of this systematic review is to assess the evidence behind the dietary requirement of protein and to assess the health effects of varying protein intake in healthy elderly persons in order to evaluate the evidence for an optimal protein intake. The literature search covered year 2000–2011. Prospective cohort, case–control, and intervention studies of a general healthy population in settings similar to the Nordic countries with protein intake from food-based sources were included. Out of a total of 301 abstracts, 152 full papers were identified as potentially relevant. After careful scrutiny, 23 papers were quality graded as A (highest, n=1, B (n=18, or C (n=4. The grade of evidence was classified as convincing, probable, suggestive, or inconclusive. The evidence is assessed as: probable for an estimated average requirement (EAR of 0.66 g good-quality protein/kg body weight (BW/day based on nitrogen balance (N-balance studies and the subsequent recommended dietary allowance (RDA of 0.83 g good-quality protein/kg BW/day representing the minimum dietary protein needs of virtually all healthy elderly persons. Regarding the optimal level of protein related to functional outcomes like maintenance of bone mass, muscle mass, and strength, as well as for morbidity and mortality, the evidence is ranging from suggestive to inconclusive. Results from particularly prospective cohort studies suggest a safe intake of up to at least 1.2–1.5 g protein/kg BW/day or approximately 15–20 E%. Overall, many of the included prospective cohort studies were difficult to fully evaluate since results mainly were obtained by food frequency questionnaires that were flawed by underreported intakes, although some studies were ‘calibrated’ to correct for under- or over-reporting. In conclusion, the evidence is assessed as probable regarding the EAR based on N-balance studies and suggestive to inconclusive regarding an optimal protein intake higher than

  10. N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, a molecule for radiohalogenation of proteins and peptides

    DEFF Research Database (Denmark)

    Aneheim, Emma; Foreman, Mark R StJ; Jensen, Holger

    2015-01-01

    In this work a new coupling reagent, N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, for radiohalogenation has been synthesized and characterized. The reagent is intended to either be attached to reduced disulfide bridges of proteins (making the halogenation site-specific) or to free termin...

  11. The Mechanism of Action of Unique Small Molecules that Inhibit the Pim Protein Kinase Blocking Prostate Cancer Cell Growth

    Science.gov (United States)

    2010-05-01

    TKO∕EV) or Pim-3 (TKO∕Pim-3). Values ( cpm ∕mg protein) are the average of three independent measurements, and the standard deviation for the mean is...article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 27, 2011

  12. Relation between Protein Intrinsic Normal Mode Weights and Pre-Existing Conformer Populations.

    Science.gov (United States)

    Ozgur, Beytullah; Ozdemir, E Sila; Gursoy, Attila; Keskin, Ozlem

    2017-04-20

    Intrinsic fluctuations of a protein enable it to sample a large repertoire of conformers including the open and closed forms. These distinct forms of the protein called conformational substates pre-exist together in equilibrium as an ensemble independent from its ligands. The role of ligand might be simply to alter the equilibrium toward the most appropriate form for binding. Normal mode analysis is proved to be useful in identifying the directions of conformational changes between substates. In this study, we demonstrate that the ratios of normalized weights of a few normal modes driving the protein between its substates can give insights about the ratios of kinetic conversion rates of the substates, although a direct relation between the eigenvalues and kinetic conversion rates or populations of each substate could not be observed. The correlation between the normalized mode weight ratios and the kinetic rate ratios is around 83% on a set of 11 non-enzyme proteins and around 59% on a set of 17 enzymes. The results are suggestive that mode motions carry intrinsic relations with thermodynamics and kinetics of the proteins.

  13. The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

    Science.gov (United States)

    Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

    2016-01-08

    Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Molecule nanoweaver

    Science.gov (United States)

    Gerald, II; Rex, E [Brookfield, IL; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL; Diaz, Rocio [Chicago, IL; Vukovic, Lela [Westchester, IL

    2009-03-10

    A method, apparatus, and system for constructing uniform macroscopic films with tailored geometric assemblies of molecules on the nanometer scale. The method, apparatus, and system include providing starting molecules of selected character, applying one or more force fields to the molecules to cause them to order and condense with NMR spectra and images being used to monitor progress in creating the desired geometrical assembly and functionality of molecules that comprise the films.

  15. CD147: a small molecule transporter ancillary protein at the crossroad of multiple hallmarks of cancer and metabolic reprogramming.

    Science.gov (United States)

    Kendrick, Agnieszka A; Schafer, Johnathon; Dzieciatkowska, Monika; Nemkov, Travis; D'Alessandro, Angelo; Neelakantan, Deepika; Ford, Heide L; Pearson, Chad G; Weekes, Colin D; Hansen, Kirk C; Eisenmesser, Elan Z

    2017-01-24

    Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner's proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.

  16. Novel association of soluble intercellular adhesion molecule 1 and soluble P-selectin with the ABO blood group in a Chinese population.

    Science.gov (United States)

    Zhang, Wenjing; Xu, Qun; Zhuang, Yunlong; Chen, Yuanfeng

    2016-08-01

    Recent studies have reported that the ABO gene can affect circulating expression levels of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble P-selectin (sP-selectin) in Caucasians. However, several factors may affect the association, including the distribution and variations of the ABO gene, ethnic diversity and the inflammatory response status. The aim of the present study was to investigate this issue in Asian subjects of various blood groups. A total of 800 blood samples were randomly selected from healthy blood donors. The ABO blood groups were examined using standard serological tests, and ABO genotypes of group A and group AB specimens were analyzed. Plasma concentrations of sICAM-1 and sP-selectin were detected by standard enzyme-linked immunosorbent assays. In healthy Chinese individuals, blood group A was detected to be significantly associated with lower circulating expression levels of sICAM-1 and sP-selectin, compared with group O. Individuals with ≥1 A1 allele had significantly lower expression levels of sICAM-1 and sP-selectin compared with all other ABO groups. The data indicate the significant association of ABO blood group antigens with sICAM-1 and sP-selectin expression levels in a healthy Chinese population, independent of the specific variations and distributions of ABO blood groups among ethnic populations. This result provides evidence for the previously unidentified role of ABO blood group antigens in the regulation of the inflammatory adhesion process. Accordingly, it can be proposed that ABO blood groups may require consideration when soluble adhesion molecules are identified as predictors for cardiovascular disease.

  17. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    Directory of Open Access Journals (Sweden)

    Sarah L Appleby

    Full Text Available Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+ population of non-adherent endothelial forming cells (naEFCs which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38 together with mature endothelial cell markers (VEGFR2, CD144 and CD31. These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8 or myeloid markers (CD11b and CD14 which distinguishes them from 'early' endothelial progenitor cells (EPCs. Functional studies demonstrated that these naEFCs (i bound Ulex europaeus lectin, (ii demonstrated acetylated-low density lipoprotein uptake, (iii increased vascular cell adhesion molecule (VCAM-1 surface expression in response to tumor necrosis factor and (iv in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs. Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  18. Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

    Science.gov (United States)

    Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

  19. A single molecule approach for measuring the transport properties and energetics of membrane proteins in heterogeneous planar bio-mimetic assemblies

    Science.gov (United States)

    Poudel, Kumud Raj

    The significance of transmembrane protein research is well documented. Numerous studies have clearly established the biological, biophysical and pharmaceutical importance that these membrane components serve. Communications through receptors regulate countless body functions and they also provide structural support to the cell. However, a lack of high-resolution structure data has limited our understanding of these proteins that make it necessary to study them in in-vitro platforms or 'bio-mimetic' assemblies. Albeit that an assortment of platforms have been suggested for in-vitro studies, the issues, however, remain the same. The lack of mobility of the proteins in artificial environments, the question of functionality that arises with mobility and the search in general for the best assembly, is still a work in progress. In this work, we have taken some of the most accepted platforms in the field and characterized them through the lens of single molecule spectroscopy. We have addressed the question of mobility by reducing it down to a single molecule and comparing it with the bulk. By utilizing the Serotonin Receptor 5HT3A we have shown that techniques such as passivation of the substrates in the assemblies by Bovine Serum Albumin has a significant effect at the molecular level. The larger size of the intracellular domain for the 5HT3A served as a great probe to understand and evaluate the interaction of a surface passivator with the integrated membrane protein. We have also taken this a step further by developing a novel, single cushion 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) assembly and added another degree of complexity- through a phase transition. We have utilized phase transition to get an insight into the local protein environment, activation energies, heterogeneity and diffusion characteristics by using Annexin V as our probe. The work presented here studies two completely different biological platforms using two entirely different transmembrane

  20. Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand.

    Science.gov (United States)

    Pattaradilokrat, Sittiporn; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Siripoon, Napaporn; Harnyuttanakorn, Pongchai

    2016-10-21

    An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown. The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima's D, Fu and Li D* and Fu and Li' F* tests) to determine any signature of selection. The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia. This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.

  1. Development of entomotoxic molecules as control agents: illustration of some protein potential uses and limits of lectins (Review

    Directory of Open Access Journals (Sweden)

    Jaber, K.

    2010-01-01

    Full Text Available Worldwide use of pesticide has dramatically increased during the last two decades, which are known to cause a lot of problems to both environment and human health. Due to the harmful effects of insecticide on environment, human health, non target organism, water pollution and increasing insect resistance, recent efforts have been made to reduce broad spectrum of toxicant added to the environment. It is necessary to balance agricultural needs with environmental and health issues when using insecticides. Currently, the use of entomotoxic proteins has been increased because of the growing need to obtain better food quality and to protect the environment. Bt toxins derived from bacteria, Bacillus thuringiensis (Bt the main commercial genetically modified organism (GMO, or entomotoxic product were successfully used against a range of insect orders such as Lepidoptera, Coleoptera and Diptera. In recent years, several investigations were focused on other entomotoxic potential, including products based on some defense proteins present in plants. Among them some classes of proteins such as digestive enzyme inhibitors and lectins showed greater potential for exploitation in transgenic-based pest control strategies. Currently, some lectins from plant and fungal origin were successfully used against a wide range of important insect pests.

  2. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    Science.gov (United States)

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  3. Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

    Science.gov (United States)

    Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

    2010-05-05

    Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  4. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    2010-05-01

    Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  5. irGPU.proton.Net: Irregular strong charge interaction networks of protonatable groups in protein molecules--a GPU solver using the fast multipole method and statistical thermodynamics.

    Science.gov (United States)

    Kantardjiev, Alexander A

    2015-04-05

    A cluster of strongly interacting ionization groups in protein molecules with irregular ionization behavior is suggestive for specific structure-function relationship. However, their computational treatment is unconventional (e.g., lack of convergence in naive self-consistent iterative algorithm). The stringent evaluation requires evaluation of Boltzmann averaged statistical mechanics sums and electrostatic energy estimation for each microstate. irGPU: Irregular strong interactions in proteins--a GPU solver is novel solution to a versatile problem in protein biophysics--atypical protonation behavior of coupled groups. The computational severity of the problem is alleviated by parallelization (via GPU kernels) which is applied for the electrostatic interaction evaluation (including explicit electrostatics via the fast multipole method) as well as statistical mechanics sums (partition function) estimation. Special attention is given to the ease of the service and encapsulation of theoretical details without sacrificing rigor of computational procedures. irGPU is not just a solution-in-principle but a promising practical application with potential to entice community into deeper understanding of principles governing biomolecule mechanisms. © 2015 Wiley Periodicals, Inc.

  6. Proposal for a scheme to generate 10 TW-Level femtosecond X-ray pulses for imaging single protein molecules at the European XFEL

    Energy Technology Data Exchange (ETDEWEB)

    Serkez, Svitozar; Kocharyan, Vitali; Saldin, Evgeni; Zagorodnov, Igor [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Geloni, Gianluca [European XFEL GmbH, Hamburg (Germany); Yefanov, Oleksander [Center for Free-Electron Laser Science, Hamburg (Germany)

    2013-06-15

    Single biomolecular imaging using XFEL radiation is an emerging method for protein structure determination using the ''diffraction before destruction'' method at near atomic resolution. Crucial parameters for such bio-imaging experiments are photon energy range, peak power, pulse duration, and transverse coherence. The largest diffraction signals are achieved at the longest wavelength that supports a given resolution, which should be better than 0.3 nm. We propose a configuration which combines self-seeding and undulator tapering techniques with the emittance-spoiler method in order to increase the XFEL output peak power and to shorten the pulse duration up to a level sufficient for performing bio-imaging of single protein molecules at the optimal photon energy range, i.e. around 4 keV. Experiments at the LCLS confirmed the feasibility of these three new techniques. Based on start-to-end simulations we demonstrate that self-seeding, combined with undulator tapering, allows one to achieve up to a 100-fold increase in peak-power. A slotted foil in the last bunch compressor is added for X-ray pulse duration control. Simulations indicate that one can achieve diffraction to the desired resolution with 50 mJ (corresponding to 10{sup 14} photons) per 10 fs pulse at 3.5 keV photon energy in a 100 nm focus. This result is exemplified using the photosystem I membrane protein as a case study.

  7. Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 protein.

    Science.gov (United States)

    Petty, Tom J; Nishimura, Taisuke; Emamzadah, Soheila; Gabus, Caroline; Paszkowski, Jerzy; Halazonetis, Thanos D; Thore, Stéphane

    2010-08-01

    Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to approximately 3.2 A resolution. They belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a=85.64, c=292.74 A. Structure determination is ongoing.

  8. Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein

    Science.gov (United States)

    Petty, Tom J.; Nishimura, Taisuke; Emamzadah, Soheila; Gabus, Caroline; Paszkowski, Jerzy; Halazonetis, Thanos D.; Thore, Stéphane

    2010-01-01

    Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing. PMID:20693667

  9. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor

    Science.gov (United States)

    Kong, Li; Zhao, Yun-Peng; Tian, Qing-Yun; Feng, Jian-Quan; Kobayashi, Tatsuya; Merregaert, Joseph; Liu, Chuan-Ju

    2016-01-01

    Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone–related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo. In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.—Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor. PMID:27075243

  10. Effect of perfluorohexane on the expression of cellular adhesion molecules and surfactant protein A in human mesothelial cells in vitro.

    Science.gov (United States)

    Haufe, Dirk; Dahmen, Klaus G; Tiebel, Oliver; Hübler, Matthias; Koch, Thea

    2011-08-01

    The intraperitoneal instillation of perfluorocarbons augmented systemic oxygenation and was protective in mesenteric ischemia-reperfusion and experimental lung injury. To study biocompatibility and potential anti-inflammatory effects of intraperitoneal perfluorocarbons, we evaluated the influence of perfluorohexane and/or inflammatory stimuli on human mesothelial cells in vitro. Perfluorohexane exposure neither impaired cell viability nor induced cellular activation. TNFα enhanced ICAM-1 expression, which was not attenuated by simultaneous perfluorohexane treatment. Concentration of intracellular surfactant protein A tended to be higher in perfluorohexane treated cells compared to controls. Our in vitro data add further evidence that intraperitoneal perfluorocarbon application is feasible without adverse local effects.

  11. LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

    Science.gov (United States)

    Oliveira, Tatiane R; Longhi, Mariana T; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-03-01

    The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. Copyright 2009 Elsevier Masson SAS. All rights reserved.

  12. Food protein-induced enterocolitis syndrome in Australia: A population-based study, 2012-2014.

    Science.gov (United States)

    Mehr, Sam; Frith, Katie; Barnes, Elizabeth H; Campbell, Dianne E

    2017-11-01

    Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated gastrointestinal allergic disorder. Large population-based FPIES studies are lacking. We sought to determine the incidence and clinical characteristics of FPIES in Australian infants. An Australia-wide survey (2012-2014) was undertaken through the Australian Paediatric Surveillance Unit, with monthly notification of new cases of acute FPIES in infants aged less than 24 months by 1400 participating pediatricians. Two hundred thirty infants with FPIES were identified. The incidence of FPIES in Australian infants (disease and FPIES to fruits, vegetables, or both. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  13. Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance.

    Science.gov (United States)

    Hamamoto, Ryo; Ito, Hidemi; Hirohara, Makoto; Chang, Ryongsok; Hongo-Hirasaki, Tomoko; Hayashi, Tomohiro

    2018-03-01

    Membrane fouling commonly occurs in all filter types during virus filtration in protein-based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose-based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post-adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose-based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379-386, 2018. © 2017 American Institute of Chemical Engineers.

  14. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

    Science.gov (United States)

    Hetrick, Byron; Han, Min Suk; Helgeson, Luke A; Nolen, Brad J

    2013-05-23

    Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

    DEFF Research Database (Denmark)

    Chi, Qijin; Farver, O; Ulstrup, Jens

    2005-01-01

    on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on/off current ratio of approximate to 9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative......A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi...... constants display tunneling features with distance-decay factors of 0.83 and 0.91 angstrom(-1) in H2O and D2O, respectively. Redox-gated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence...

  16. Dynamics of liquids, molecules, and proteins measured with ultrafast 2D IR vibrational echo chemical exchange spectroscopy.

    Science.gov (United States)

    Fayer, M D

    2009-01-01

    A wide variety of molecular systems undergo fast structural changes under thermal equilibrium conditions. Such transformations are involved in a vast array of chemical problems. Experimentally measuring equilibrium dynamics is a challenging problem that is at the forefront of chemical research. This review describes ultrafast 2D IR vibrational echo chemical exchange experiments and applies them to several types of molecular systems. The formation and dissociation of organic solute-solvent complexes are directly observed. The dissociation times of 13 complexes, ranging from 4 ps to 140 ps, are shown to obey a relationship that depends on the complex's formation enthalpy. The rate of rotational gauche-trans isomerization around a carbon-carbon single bond is determined for a substituted ethane at room temperature in a low viscosity solvent. The results are used to obtain an approximate isomerization rate for ethane. Finally, the time dependence of a well-defined single structural transformation of a protein is measured.

  17. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

    Directory of Open Access Journals (Sweden)

    Yaffa Mizrachi Nebenzahl

    Full Text Available In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

  18. Molecule Matters

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 14; Issue 4. Molecule Matters – van der Waals Molecules - History and Some Perspectives on Intermolecular Forces. E Arunan. Feature Article Volume 14 Issue 4 April 2009 pp 346-356 ...

  19. Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Ruodan Xu

    2015-04-01

    Full Text Available Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES cell lineage expressing enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain (α-MHC promoter (pαMHC-EGFP, we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3 on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to

  20. PoSSuM v.2.0: data update and a new function for investigating ligand analogs and target proteins of small-molecule drugs.

    Science.gov (United States)

    Ito, Jun-ichi; Ikeda, Kazuyoshi; Yamada, Kazunori; Mizuguchi, Kenji; Tomii, Kentaro

    2015-01-01

    PoSSuM (http://possum.cbrc.jp/PoSSuM/) is a database for detecting similar small-molecule binding sites on proteins. Since its initial release in 2011, PoSSuM has grown to provide information related to 49 million pairs of similar binding sites discovered among 5.5 million known and putative binding sites. This enlargement of the database is expected to enhance opportunities for biological and pharmaceutical applications, such as predictions of new functions and drug discovery. In this release, we have provided a new service named PoSSuM drug search (PoSSuMds) at http://possum.cbrc.jp/PoSSuM/drug_search/, in which we selected 194 approved drug compounds retrieved from ChEMBL, and detected their known binding pockets and pockets that are similar to them. Users can access and download all of the search results via a new web interface, which is useful for finding ligand analogs as well as potential target proteins. Furthermore, PoSSuMds enables users to explore the binding pocket universe within PoSSuM. Additionally, we have improved the web interface with new functions, including sortable tables and a viewer for visualizing and downloading superimposed pockets. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

    Science.gov (United States)

    Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

    2016-05-01

    G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

    International Nuclear Information System (INIS)

    Funk, W.D.; MacGillivray, R.T.A.; Mason, A.B.; Brown, S.A.; Woodworth, R.C.

    1990-01-01

    A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO 4 -polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19 F-Tyr hTF/2N gave four well-resolved 19 F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

  3. Effects of Dietary Supplementation with Hainanmycin on Protein Degradation and Populations of Ammonia-producing Bacteria

    Directory of Open Access Journals (Sweden)

    Z. B. Wang

    2013-05-01

    Full Text Available An in vitro fermentation was conducted to determine the effects of hainanmycin on protein degradation and populations of ammonia-producing bacteria. The substrates (DM basis for in vitro fermentation consisted of alfalfa hay (31.7%, Chinese wild rye grass hay (28.3%, ground corn grain (24.5%, soybean meal (15.5% with a forage: concentrate of 60:40. Treatments were the control (no additive and hainanmycin supplemented at 0.1 (H0.1, 1 (H1, 10 (H10, and 100 mg/kg (H100 of the substrates. After 24 h of fermentation, the highest addition level of hainanmycin decreased total VFA concentration and increased the final pH. The high addition level of hainanmycin (H1, H10, and H100 reduced (p0.05. After 24 h of fermentation, H10 and H100 increased (p<0.05 concentrations of peptide nitrogen and AA nitrogen and proteinase activity, and decreased (p<0.05 NH3-N concentration and deaminase activity compared with control. Peptidase activitives were not affected by hainanmycin. Hainanmycin supplementation only inhibited the growth of Butyrivibrio fibrisolvens, which is one of the species of low deaminative activity. Hainanmycin supplementation also decreased (p<0.05 relative population sizes of hyper-ammonia-producing species, except for H0.1 on Clostridium aminophilum. It was concluded that dietary supplementation with hainanmycin could improve ruminal fermentation and modify protein degradation by changing population size of ammonia-producing bacteria in vitro; and the addition level of 10 mg/kg appeared to achieve the best results.

  4. Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

    Science.gov (United States)

    Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

    2012-12-15

    and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.

  5. An improved model of radiative transfer for the NLTE problem in the NIR bands of CO2 and CO molecules in the daytime atmosphere of Mars. 2. Population of vibrational states

    Science.gov (United States)

    Ogibalov, V. P.; Shved, G. M.

    2017-09-01

    The near-infrared (NIR) emission of the Martian atmosphere in the CO2 bands at 4.3, 2.7, 2.0, 1.6, 1.4, 1.3, 1.2, and 1.05 µm and in the CO bands at 4.7, 2.3, 1.6, and 1.2 µm is mainly generated under nonlocal thermodynamic equilibrium (NLTE) conditions for vibrational states, the transitions from which form the specified bands. The paper presents the results of simulations of the population of these states under NLTE for daytime conditions. In the cold high-latitude troposphere, the NLTE takes place much lower than in the troposphere under typical temperature conditions. If the NIR-radiation reflection from the surface is ignored, the population of high vibrational states substantially decreases, at least, in some layer of the lower atmosphere. However, inelastic collisions of CO2 and CO molecules with O atoms produce no considerable influence on the values of populations. The population of vibrational states, the transitions from which form NIR bands, is also almost insensitive to possible large values of the quenching-in-collision rate constants of vibrational states higher than CO2(0001). However, very large errors in the estimates of the population of vibrational states of the CO2 molecule (rather than the CO molecule!) can be caused by the uncertainty in the values of the rate constant of exchange between CO2 molecules by the energy quantum of the asymmetric stretching vibrational mode. For this intermolecular exchange, we recommend a possible way to restrict the vibrational excitation degree of the molecule that is a collision partner and to maintain simultaneously a sufficiently high accuracy in the population estimate.

  6. MoMa: From Molecules to Man: Space Research Applied to the improvement of the Quality of Life of the Ageing Population on Earth. Evolution of a project

    Science.gov (United States)

    Zambito, Anna Maria; Curcio, Francesco; Meli, Antonella; Saverio Ambesi-Impiombato, Francesco

    The "MoMa" project: "From Molecules to Man: Space Research Applied to the improvement of the Quality of Life of the Ageing Population on Earth started June 16 2006 and finished right on schedule June 25 2009, has been the biggest of the three projects funded by ASI in the sector "Medicine and Biotechnology. In the last years the scientific community had formed a national chain of biomedical spatial research with different research areas. MoMa responds to the necessity of unification in ASI of the two areas "Radiobiology and Protection" and "Cellular and Molecular Biotechnology" in a line of joint research: "Biotechnological Applications" were the interests of all groups would be combined and unified in a goal of social relevance. MoMa is the largest project ever developed in the biomedical area in Italy, the idea was born thinking about the phenomenon of acceleration of the aging process observed in space, and already described in literature, and the aim of studying the effects of the space environment at cellular, molecular and human organism level. "MoMa" was divided into three primary areas of study: Molecules, Cells and Man with an industrial area alongside. This allowed to optimize the work and information flows within the scientific research more similar and more culturally homogeneous and allowed a perfect industrial integration in a project of great scientific importance. Within three scientific areas 10 scientific lines in total are identified, each of them coordinated by a subcontractor. The rapid and efficient exchange of information between different areas of science and the development of industrial applications in various areas of interest have been assured by a strong work of Scientific Coordination of System Engineering and Quality Control. After three years of intense and coordinated activities within the MoMa project, the objectives achieved are very significant not only as regards the scientific results and the important hardware produced but

  7. Atkins' molecules

    CERN Document Server

    Atkins, Peters

    2003-01-01

    Originally published in 2003, this is the second edition of a title that was called 'the most beautiful chemistry book ever written'. In it, we see the molecules responsible for the experiences of our everyday life - including fabrics, drugs, plastics, explosives, detergents, fragrances, tastes, and sex. With engaging prose Peter Atkins gives a non-technical account of an incredible range of aspects of the world around us, showing unexpected connections, and giving an insight into how this amazing world can be understood in terms of the atoms and molecules from which it is built. The second edition includes dozens of extra molecules, graphical presentation, and an even more accessible and enthralling account of the molecules themselves.

  8. Interstellar Molecules

    Science.gov (United States)

    Solomon, Philip M.

    1973-01-01

    Radioastronomy reveals that clouds between the stars, once believed to consist of simple atoms, contain molecules as complex as seven atoms and may be the most massive objects in our Galaxy. (Author/DF)

  9. Single molecules and nanotechnology

    CERN Document Server

    Vogel, Horst

    2007-01-01

    This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

  10. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    Full Text Available Yixuan Zeng,1,2,* Wenyuan Guo,1,* Guangqing Xu,3 Qinmei Wang,4 Luyang Feng,1,2 Simei Long,1 Fengyin Liang,1 Yi Huang,1 Xilin Lu,1 Shichang Li,5 Jiebin Zhou,5 Jean-Marc Burgunder,6 Jiyan Pang,5 Zhong Pei1,2 1Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Disease, The First Affiliated Hospital, Sun Yat-sen University, 2Guangzhou Center, Chinese Huntington’s Disease Network, 3Department of Rehabilitation, The First Affiliated Hospital, 4Key laboratory on Assisted Circulation, Ministry of Health, Department of Cardiovascular Medicine of the First Affiliated Hospital, 5School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 6Swiss Huntington’s Disease Center, Department of Neurology, University of Bern, Bern, Switzerland *These authors contributed equally to this work Abstract: Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt. Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a

  11. Effects of different protein concentrations on longevity and feeding behavior of two adult populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae)

    International Nuclear Information System (INIS)

    Placido-Silva, Maria do Carmo; Silva Neto, Alberto M. da; Joachim-Bravo, Iara S.; Zucoloto, Fernando S.

    2006-01-01

    The effects of protein intake on two adult male and female populations of Ceratitis capitata Wiedemann were assessed. One population consisted of flies reared for twenty years in the laboratory (Lab-pop); the other population consisted both of flies reared in the laboratory for approximately fifteen years and of the periodically introduced wild flies (Hybrid-pop). Three diets were tested: a no-yeast diet and two diets containing yeast (protein source) at the concentrations 6.5 g or 1.5 g per 100 ml diet. The parameters analyzed were: adult longevity, diet intake with and without yeast, and discrimination threshold for yeast. Protein intake increased Lab-pop adult longevity and did not affect longevity of the Hybrid-pop. Longevity in each population was similar for males and females fed on the same diet. Food behavior were similar for male and female adults of both populations; all preferred diets containing protein (yeast). Males and females in both populations ingested similar amounts of each diet. The discrimination threshold for yeast was similar for all males (0.5 g yeast/100 ml diet); Lab-pop females were able to detect the presence of smaller quantities of yeast in their diet, thus having a higher discrimination capacity (0.4 g/100 ml diet) as compared to the Hybrid-pop females (0.6 g/ 100 ml diet). (author)

  12. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows.

    Science.gov (United States)

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-06-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (Pdairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat stress in dairy cows in the future.

  13. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Halawani Dalia

    2007-10-01

    Full Text Available Abstract Background HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER-associated protein degradation (ERAD. Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub-proteasome degradative system through an interaction with β-TrCP, a component of the SCFβ-TrCP E3 Ub ligase complex. Results Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFβ-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. Conclusion Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFβ-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

  14. Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

    Science.gov (United States)

    Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

    2015-01-01

    Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain's neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case–control and review studies were considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory. PMID:26445635

  15. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows*

    Science.gov (United States)

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-01-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (Pheat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat stress in dairy cows in the future. PMID:26055916

  16. Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K alpha-Helix

    DEFF Research Database (Denmark)

    Sivaramakrishnan, S.; Sung, J.; Ali, M.

    2009-01-01

    as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantity this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations...

  17. Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis

    Czech Academy of Sciences Publication Activity Database

    Harris, N.; Koppel, J.; Zsila, F.; Juhás, Štefan; Ilková, G.; Kogan, F. Y.; Lahmy, O.; Wildbaum, G.; Karin, N.; Zhuk, R.; Gregor, P.

    2016-01-01

    Roč. 65, č. 4 (2016), s. 285-294 ISSN 1023-3830 Institutional support: RVO:67985904 Keywords : small molecule drug * glycosaminoglycan * heparin binding protein * heparan sulfate * inflammation * autoimmune disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.659, year: 2016

  18. Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

    Science.gov (United States)

    Maier, Richard H; Maier, Christina J; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2012-12-01

    Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Adhesion molecules

    CERN Document Server

    Preedy, Victor R

    2016-01-01

    This book covers the structure and classification of adhesion molecules in relation to signaling pathways and gene expression. It discusses immunohistochemical localization, neutrophil migration, and junctional, functional, and inflammatory adhesion molecules in pathologies such as leukocyte decompression sickness and ischemia reperfusion injury. Highlighting the medical applications of current research, chapters cover diabetes, obesity, and metabolic syndrome; hypoxia; kidney disease; smoking, atrial fibrillation, and heart disease, the brain and dementia; and tumor proliferation. Finally, it looks at molecular imaging and bioinformatics, high-throughput technologies, and chemotherapy.

  20. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Hussain, Shah; Pezzei, Cornelia [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Güzel, Yüksel [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria); Rainer, Matthias [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Huck, Christian W., E-mail: Christian.W.Huck@uibk.ac.at [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Bonn, Günther K. [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria)

    2014-12-10

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  1. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    International Nuclear Information System (INIS)

    Hussain, Shah; Pezzei, Cornelia; Güzel, Yüksel; Rainer, Matthias; Huck, Christian W.; Bonn, Günther K.

    2014-01-01

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  2. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  3. Correlates of High Serum C-Reactive Protein Levels in a Socioeconomically Disadvantaged Population

    Directory of Open Access Journals (Sweden)

    Xianglan Zhang

    2008-01-01

    Full Text Available Individuals from low socioeconomic backgrounds are disproportionately affected by the burden of cardiovascular disease (CVD, yet data regarding risk factors in this population are lacking, particularly regarding emerging biomarkers of CVD such as C-reactive protein (CRP. We measured high-sensitivity CRP and examined its association with demographic and lifestyle factors in a sample of 792 participants aged 40–79 years from the Southern Community Cohort Study, which has an over-representation of socioeconomically disadvantaged individuals (over 60% with a total annual household income 3 mg/L varied significantly by sex, race, smoking status, and body mass index (BMI. The multivariable-adjusted prevalence odds ratios (ORs (95% CIs for having elevated CRP were 1.6 (1.1–2.3 for women vs. men, 1.4 (0.9–2.0 for African Americans vs. whites, 2.3 (1.4–3.8 for African American women vs. white men, 1.8 (1.2–2.7 for current smokers vs. non-smokers, and 4.2 (2.7–6.6 for obese (BMI 30.0–44.9 kg/m2 vs. healthy-weight (BMI 18.3–24.9 kg/m2 participants. Further stratified analyses revealed that the association between BMI and elevated CRP was stronger among African Americans than whites and women than men, with prevalence ORs (95% CI comparing obese vs. healthy-weight categories reaching 22.8 (7.1–73.8 for African American women. In conclusion, in this socioeconomically disadvantaged population, sex, race, smoking, and BMI were associated with elevated CRP. Moreover, inflammatory response to obesity differed by race and sex, which may contribute to CVD disparities.

  4. C-reactive protein, APOE genotype and longitudinal cognitive change in an older population

    Science.gov (United States)

    Lima, Thomas A. S.; Adler, Amanda L.; Minett, Thais; Matthews, Fiona E.; Brayne, Carol; Marioni, Riccardo E.

    2014-01-01

    Background: circulating measures of inflammatory markers, such as C-reactive protein (CRP) have been associated with an increased risk of future cognitive decline. However, the nature of the relationship among the very old (>75 years) is unclear. Cross-sectional evidence suggests that elevated CRP may even be protective in this age group. This study examines these associations longitudinally. Methods: logistic regression was used to investigate the association between CRP and drop in cognitive performance (≥3 point change on the Mini-Mental State Examination) over a 4-year period in a population of 266 people, mean age 77 years. Results: increased levels of CRP were associated with a decreased risk of a drop in cognitive performance; however, this association was only seen in those without an APOE e4 allele [odds ratio of decline per unit increase in ln(CRP) 0.57, P = 0.04]. The magnitude of the finding remained consistent after adjustment for cardiovascular confounders (smoking, drinking, MI, stroke, diabetes, education, medication and blood pressure). For those with an e4 allele, the relationship with longitudinal cognitive decline was neither statistically significant nor in a consistent direction after controlling for acute inflammation. Conclusions: this study strengthens previous cross-sectional findings and shows elevated levels of CRP to be linked to a decreased risk of longitudinal cognitive decline in the very old. However, as with prior analyses, this was only observed in those not carrying an APOE e4 allele. Future work on larger APOE e4 allele carrying samples is required to determine the nature of the association in this population. PMID:24305621

  5. Convergence of a diabetes mellitus, protein energy malnutrition, and TB epidemic: the neglected elderly population.

    Science.gov (United States)

    Menon, Sonia; Rossi, Rodolfo; Nshimyumukiza, Leon; Wusiman, Aibibula; Zdraveska, Natasha; Eldin, Manal Shams

    2016-07-26

    On a global scale, nearly two billion persons are infected with Mycobacterium tuberculosis. From this vast reservoir of latent tuberculosis (TB) infection, a substantial number will develop active TB during their lifetime, with some being able to transmit TB or Multi-drug- resistant (MDR) TB to others. There is clinical evidence pointing to a higher prevalence of infectious diseases including TB among individuals with Diabetes Mellitus (DM). Furthermore, ageing and diabetes mellitus may further aggravate protein-energy malnutrition (PEM), which in turn impairs T-lymphocyte mediated immunologic defenses, thereby increasing the risk of developing active TB and compromising TB treatment. This article aims to a) highlight synergistic mechanisms associated with immunosenescence, DM and PEM in relation to the development of active TB and b) identify nutritional, clinical and epidemiological research gaps. To explore the synergistic relationship between ageing, DM, tuberculosis and PEM, a comprehensive review was undertaken. The MEDLINE and the Google Scholar databases were searched for articles published from 1990 to March 2015, using different MESH keywords in various combinations. Ageing and DM act synergistically to reduce levels of interferon gamma (IFN- γ), thereby increasing susceptibility to TB, for which cell mediated immunity (CMI) plays an instrumental role. These processes can set in motion a vicious nutritional cycle which can predispose to PEM, further impairing the CMI and consequently limiting host defenses. This ultimately transforms the latent TB infection into active disease. A clinical diagnostic algorithm and clinical guidelines need to be established for this population. Given the increase in ageing population with DM and PEM, especially in resource-poor settings, these synergistic tripartite interactions must be examined if a burgeoning TB epidemic is to be averted. Implementation of a comprehensive, all-encompassing approach to curb transmission

  6. Molecule Matters

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 16; Issue 12. Molecule Matters - Dinitrogen. A G Samuelson J Jabadurai. Volume 16 Issue 12 ... Author Affiliations. A G Samuelson1 J Jabadurai1. Department of Inroganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India.

  7. Molecule Matters

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 11; Issue 9. Molecule Matters - A Chromium Compound with a Quintuple Bond. K C Kumara Swamy. Feature Article Volume 11 Issue 9 September 2006 pp 72-75. Fulltext. Click here to view fulltext PDF. Permanent link:

  8. Serological diagnosis for active tuberculosis in Malaysian population: Comparison of four protein candidate

    Directory of Open Access Journals (Sweden)

    Siti Suraiya

    2012-05-01

    Full Text Available Objective: To asses the ability of 4 types of Mtb proteins-ESAT6, SCWP, MAN and Ag85 to serve as indicator for active tuberculosis among Malaysian population. Methods: Sera from 90 individuals, 60 from confirmed pulmonary tuberculosis patients and 30 healthy PPD negative individuals were tested for presence of anti-IgG and anti IgA by ELISA assay. Result: Mean concentration of IgG and IgA were higher in patients compared to healthy Positivity of the ELISA test were calculated, taking the cut off value at mean +2 SD of healthy sera. The sensitivity of the ELISA IgA assay for ESAT 6, SCWP, MAN and Ag85 were 81.1%, 83.3%, 11.7% and 53.3% respectively. The sensitivity of the ELISA IgG asay for ESAT 6, SCWP, MAN and Ag85 were 71.0%, 71.0%, 71.0% and 21.0% respectively. Conclusion: Detectionof IgA against SCWP promised a good indicator for active tuberculosis infection among Malaysian.

  9. Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

    Science.gov (United States)

    Pandžić, Elvis; Abu-Arish, Asmahan; Whan, Renee M; Hanrahan, John W; Wiseman, Paul W

    2018-02-16

    Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Science.gov (United States)

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  11. Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

    Science.gov (United States)

    Latour, Chloé; Besson-Fournier, Céline; Meynard, Delphine; Silvestri, Laura; Gourbeyre, Ophélie; Aguilar-Martinez, Patricia; Schmidt, Paul J; Fleming, Mark D; Roth, Marie-Paule; Coppin, Hélène

    2016-01-01

    Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and β2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice. © 2015 by the American Association for the Study of Liver Diseases.

  12. Functional cross-talk between the cellular prion protein and the neural cell adhesion molecule is critical for neuronal differentiation of neural stem/precursor cells.

    Science.gov (United States)

    Prodromidou, Kanella; Papastefanaki, Florentia; Sklaviadis, Theodoros; Matsas, Rebecca

    2014-06-01

    Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP-NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild-type and PrP null (-/-) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM-induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin-positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP-/- mice compared with PrP+/+ mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5-bromo-2'-deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP-/- mice. Analysis of the migration of SVZ cells in microexplant cultures from wild-type and PrP-/- mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM-dependent. © 2014 AlphaMed Press.

  13. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  14. WDL-RF: Predicting Bioactivities of Ligand Molecules Acting with G Protein-coupled Receptors by Combining Weighted Deep Learning and Random Forest.

    Science.gov (United States)

    Wu, Jiansheng; Zhang, Qiuming; Wu, Weijian; Pang, Tao; Hu, Haifeng; Chan, Wallace K B; Ke, Xiaoyan; Zhang, Yang; Wren, Jonathan

    2018-02-08

    Precise assessment of ligand bioactivities (including IC50, EC50, Ki, Kd, etc.) is essential for virtual screening and lead compound identification. However, not all ligands have experimentally-determined activities. In particular, many G protein-coupled receptors (GPCRs), which are the largest integral membrane protein family and represent targets of nearly 40% drugs on the market, lack published experimental data about ligand interactions. Computational methods with the ability to accurately predict the bioactivity of ligands can help efficiently address this problem. We proposed a new method, WDL-RF, using weighted deep learning and random forest, to model the bioactivity of GPCR-associated ligand molecules. The pipeline of our algorithm consists of two consecutive stages: 1) molecular fingerprint generation through a new weighted deep learning method, and 2) bioactivity calculations with a random forest model; where one uniqueness of the approach is that the model allows end-to-end learning of prediction pipelines with input ligands being of arbitrary size. The method was tested on a set of twenty-six non-redundant GPCRs that have a high number of active ligands, each with 200∼4000 ligand associations. The results from our benchmark show that WDL-RF can generate bioactivity predictions with an average root-mean square error 1.33 and correlation coefficient (r2) 0.80 compared to the experimental measurements, which are significantly more accurate than the control predictors with different molecular fingerprints and descriptors. In particular, data-driven molecular fingerprint features, as extracted from the weighted deep learning models, can help solve deficiencies stemming from the use of traditional hand-crafted features and significantly increase the efficiency of short molecular fingerprints in virtual screening. The WDL-RF web server, as well as source codes and datasets of WDL-RF, is freely available at https://zhanglab.ccmb.med.umich.edu/WDL-RF/ for

  15. Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

    Science.gov (United States)

    Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

    2011-02-25

    FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

  16. Health effects of protein intake in healthy elderly populations: a systematic literature review

    DEFF Research Database (Denmark)

    Pedersen, Agnes N.; Cederholm, Tommy

    2014-01-01

    The purpose of this systematic review is to assess the evidence behind the dietary requirement of protein and to assess the health effects of varying protein intake in healthy elderly persons in order to evaluate the evidence for an optimal protein intake. The literature search covered year 2000-...

  17. Dietary protein and risk of hypertension in a Dutch older population: the Rotterdam Study

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.; Engberink, M.F.; Rooij, van F.J.A.; Hofman, A.; Veer, van 't P.; Witteman, J.C.M.; Geleijnse, J.M.

    2010-01-01

    AB Background: Several observational studies suggest an inverse association of protein with blood pressure (BP). However, little is known about the role of dietary protein from specific sources in BP. Method: We examined the relation between several types of dietary protein (total, plant, animal,

  18. The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

    Science.gov (United States)

    Zheng, Benle; Wu, Zaifu; Xu, Baohua

    2014-01-01

    This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

  19. Antibody reactivity against Helicobacter pylori proteins in a sample of the Spanish adult population in 2008-2013.

    Science.gov (United States)

    Fernández-de-Larrea, Nerea; Michel, Angelika; Romero, Beatriz; Butt, Julia; Pawlita, Michael; Pérez-Gómez, Beatriz; Castaño-Vinyals, Gemma; Moreno, Victor; Martín, Vicente; Amiano, Pilar; Castilla, Jesús; Fernández-Tardón, Guillermo; Dierssen-Sotos, Trinidad; Clofent, Juan; Alguacil, Juan; Huerta, José María; Jiménez-Moleón, José Juan; Barricarte, Aurelio; Molinuevo, Amaia; Fernández-Villa, Tania; Casabonne, Delphine; Sierra, Ángeles; Kogevinas, Manolis; de Sanjosé, Silvia; Pollán, Marina; Del Campo, Rosa; Waterboer, Tim; Aragonés, Nuria

    2017-10-01

    Differences in Helicobacter pylori protein expression have been related to the risk of severe gastric diseases. In Spain, a marked geographic pattern in gastric cancer mortality has long been reported. To characterize antibody reactivity patterns against 16 H. pylori proteins, by age, sex, and region of birth, in a large sample of the Spanish adult population. Antibody reactivity was quantified by H. pylori multiplex serology in a sample from the control group of the multicase-control study MCC-Spain. For this analysis, 2555 population-based controls were included. Each participant was classified as seropositive or seronegative for each protein according to specific cutoffs. Overall H. pylori seroprevalence was defined as positivity against ≥4 proteins. Descriptive analyses by age, sex, and region of birth were performed for both seroprevalence and seroreactivity (continuous measure). Differences among groups were tested by logistic and linear regression models. Overall H. pylori seroprevalence increased with age in both sexes. For ages 55-74, seroprevalence was lower in women than in men (84% vs 92%, Ppylori seropositive subjects, proteins with the highest seroprevalence were GroEL, NapA, HP231, and Omp. Seropositivity for most of the proteins increased or remained stable with age, rising mainly for CagA, GroEL, and HyuA in women. A clear cohort effect was not observed. This is the first study to describe the antibody patterns against 16 H. pylori proteins in the Spanish population. We found variability in the H. pylori antibody profiles according to both individual factors such as age and sex, and environmental factors such as the region of birth. The slightness of the reduction in seropositivity with decreasing age highlights the ongoing importance of this infection. © 2017 John Wiley & Sons Ltd.

  20. Daytime napping, sleep duration and serum C reactive protein: a population-based cohort study.

    Science.gov (United States)

    Leng, Yue; Ahmadi-Abhari, Sara; Wainwright, Nick W J; Cappuccio, Francesco P; Surtees, Paul G; Luben, Robert; Brayne, Carol; Khaw, Kay-Tee

    2014-11-11

    To explore whether daytime napping and sleep duration are linked to serum C reactive protein (CRP), a pro-inflammatory marker, in an older aged British population. Cross-sectional study. European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study. A total of 5018 men and women aged 48-92 years reported their sleep habits and had serum CRP levels measured. CRP was measured (mg/L) during 2006-2011 in fresh blood samples using high-sensitivity methods. Participants reported napping habits during 2002-2004, and reported sleep quantity during 2006-2007. Multivariable linear regression models were used to examine the association between napping and log-transformed CRP, and geometric mean CRP levels were calculated. After adjustment for age and sex, those who reported napping had 10% higher CRP levels compared with those not napping. The association was attenuated but remained borderline significant (β=0.05 (95% CI 0.00 to 0.10)) after further adjustment for social class, education, marital status, body mass index, physical activity, smoking, alcohol intake, self-reported health, pre-existing diseases, systolic blood pressure, hypnotic drug use, depression and in women-only hormone replacement therapy use. The geometric means (95% CI) of CRP levels were 2.38 (2.29 to 2.47) mg/L and 2.26 (2.21 to 2.32) mg/L for those who reported napping and no napping, respectively. A U-shaped association was observed between time spent in bed at night and CRP levels, and nighttime sleep duration was not associated with serum CRP levels. The association between napping and CRP was stronger for older participants, and among extremes of time spent in bed at night. Daytime napping was associated with increased CRP levels in an older aged British population. Further studies are needed to determine whether daytime napping is a cause for systemic inflammation, or if it is a symptom or consequence of underlying health problems. Published by the BMJ Publishing Group Limited

  1. [Effect of starvation on blood protein levels in the population of Dobrinja (1992-1995)].

    Science.gov (United States)

    Hasković, E

    2000-01-01

    In nutritional protein deficiency, numerous studies verified utilization of amino acids generated from tissue degradation in intensive protein synthesis. Unlike liver, muscle protein synthesis is extremely dependent on external supplies of essential amino acids. Prolonged nutritional protein deficiency results in decrease of body weight as well as total protein concentration, in particular in early days of starvation. In prolonged starvation during the war, significant decrease of body weight was registered in 70 subjects while their total protein concentration remained within the expected range and did not significantly differ the values recorded in the control group. Concentration of serum albumines in the control group was lower than the concentration recorded in the tested group, while the serum globulins concentration was higher in the control group. Although the difference in body weight between the tested and the control group was statistically significant, no significant difference in the concentration of total proteins, albumines and globulines was recorded.

  2. Cell-cycle protein expression in a population-based study of ovarian and endometrial cancers

    Directory of Open Access Journals (Sweden)

    Ashley S. Felix

    2015-02-01

    Full Text Available Aberrant expression of cyclin-dependent kinase (CDK inhibitors is implicated in the carcinogenesis of many cancers, including ovarian and endometrial cancers. We examined associations between CDK inhibitor expression, cancer risk factors, tumor characteristics, and survival outcomes among ovarian and endometrial cancer patients enrolled in a population-based case control study. Expression (negative vs. positive of three CDK inhibitors (p16, p21, p27 and ki67 was examined with immunohistochemical staining of tissue microarrays. Logistic regression was used to estimate adjusted odds ratios (ORs and 95% confidence intervals (CIs for associations between biomarkers, risk factors, and tumor characteristics. Survival outcomes were available for ovarian cancer patients and examined using Kaplan-Meier plots and Cox proportional hazards regression. Among ovarian cancer patients (n=175, positive p21 expression was associated with endometrioid tumors (OR=12.22, 95% CI=1.45-102.78 and higher overall survival (log-rank p=0.002. In Cox models adjusted for stage, grade, and histology, the association between p21 expression and overall survival was borderline significant (hazard ratio=0.65, 95% CI=0.42-1.05. Among endometrial cancer patients (n=289, positive p21 expression was inversely associated with age (OR ≥ 65 years of age=0.25, 95% CI=0.07-0.84 and current smoking status (OR: 0.33, 95% CI 0.15, 0.72 compared to negative expression. Our study showed heterogeneity in expression of cell-cycle proteins associated with risk factors and tumor characteristics of gynecologic cancers. Future studies to assess these markers of etiological classification and behavior may be warranted.

  3. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

    Directory of Open Access Journals (Sweden)

    Thomas L Prince

    Full Text Available The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

  4. Dietary Protein Intake in a Multi-ethnic Asian Population of Healthy Participants and Chronic Kidney Disease Patients.

    Science.gov (United States)

    Teo, Boon Wee; Toh, Qi Chun; Xu, Hui; Yang, Adonsia Y T; Lin, Tingxuan; Li, Jialiang; Lee, Evan J C

    2015-04-01

    Clinical practice guidelines recommend different levels of dietary protein intake in predialysis chronic kidney disease (CKD) patients. It is unknown how effectively these recommendations perform in a multi-ethnic Asian population, with varied cultural beliefs and diets. We assess the profi le of protein intake in a multi-ethnic Asian population, comparing healthy participants and CKD patients. We analysed the 24-hour urine collections of the Asian Kidney Disease Study (AKDS) and the Singapore Kidney Function Study (SKFS) to estimate total protein intake (TPI; g/day). We calculated ideal body weight (IDW; kg): 22.99 × height2 (m). Standard statistical tests were applied where appropriate, and linear regression was used to assess associations of continuous variables with protein intake. There were 232 CKD patients and 103 healthy participants with 35.5% diabetics. The mean TPI in healthy participants was 58.89 ± 18.42 and the mean TPI in CKD patients was 53.64 ± 19.39. By US National Kidney Foundation (NKF) guidelines, 29/232 (12.5%) of CKD patients with measured glomerular filtration rate (GFR) patients had TPI-IDW >0.75g/kg/ day. By American Dietetic Association (ADA) guidelines, 34.7% (44/127) of CKD patients with GFR patients with GFR protein intake of between 0.3 to 0.5 g/kg/day. A total of 21.9% (25/114) of diabetic CKD patients had protein intake between 0.8 to 0.9 g/kg/day. On average, the protein intake of most CKD patients exceeds the recommendations of guidelines. Diabetic CKD patients should aim to have higher protein intakes.

  5. Surfactant protein B deficiency and gene mutations for neonatal respiratory distress syndrome in China Han ethnic population

    Science.gov (United States)

    Yin, Xiaojuan; Meng, Fanping; wang, Yan; Xie, Lu; Kong, Xiangyong; Feng, Zhichun

    2013-01-01

    Objective: To determine whether the SP-B deficiency and gene mutations in exon 4 is associated with neonatal RDS in China Han ethnic population. Methods: The study population consisted of 40 neonates with RDS and 40 neonates with other diseases as control in China Han ethnic population. We Compared SP-B expression in lung tissue and bronchoalveolar lavage fluid with immunoblotting, and analyzed mutations in the SP-B gene with polymerase chain reaction (PCR) and gene sequencing. Results: In RDS group, low mature Surfactant protein B was found in both lung tissue and bronchoalveolar lavage fluid in 8 neonates. In control group, only 4 neonates with low mature Surfactant protein B in both lung tissue and bronchoalveolar lavage fluid. In RDS group, 20 neonates were found to have mutations in exon 4, 12 homozygous mutations with C/C genotype and 8 heterozygous mutations with C/T genotype in surfactant protein B gene+1580 polymorphism. There were 8 cases mutations in control group, 1 in C/C and 7 in C/T genotype. The frequency of homozygotes with C/C genotype was 0.3 and frequency of heterozygotes with C/T genotype was 0.02 in RDS group. In control group, frequency of homozygotes with C/C genotype was 0.025 and frequency of heterozygote with C/T genotype was 0.175. Conclusion: Low mature Surfactant protein B is associated with the pathogenesis of neonatal respiratory distress syndrome (RDS) in China Han ethnic population. Mutations in exon 4 of the surfactant protein B gene demonstrate an association between homozygous mutations with C/C genotype in SP-B gene and neonatal RDS. PMID:23330012

  6. Similar rates of protein adaptation in Drosophila miranda and D. melanogaster, two species with different current effective population sizes

    Directory of Open Access Journals (Sweden)

    Bachtrog Doris

    2008-12-01

    Full Text Available Abstract Background Adaptive protein evolution is common in several Drosophila species investigated. Some studies point to very weak selection operating on amino-acid mutations, with average selection intensities on the order of Nes ~ 5 in D. melanogaster and D. simulans. Species with lower effective population sizes should undergo less adaptation since they generate fewer mutations and selection is ineffective on a greater proportion of beneficial mutations. Results Here I study patterns of polymorphism and divergence at 91 X-linked loci in D. miranda, a species with a roughly 5-fold smaller effective population size than D. melanogaster. Surprisingly, I find a similar fraction of amino-acid mutations being driven to fixation by positive selection in D. miranda and D. melanogaster. Genes with higher rates of amino-acid evolution show lower levels of neutral diversity, a pattern predicted by recurrent adaptive protein evolution. I fit a hitchhiking model to patterns of polymorphism in D. miranda and D. melanogaster and estimate an order of magnitude higher selection coefficients for beneficial mutations in D. miranda. Conclusion This analysis suggests that effective population size may not be a major determinant in rates of protein adaptation. Instead, adaptation may not be mutation-limited, or the distribution of fitness effects for beneficial mutations might differ vastly between different species or populations. Alternative explanation such as biases in estimating the fraction of beneficial mutations or slightly deleterious mutation models are also discussed.

  7. Multi-Population Selective Genotyping to Identify Soybean [Glycine max (L.) Merr.] Seed Protein and Oil QTLs.

    Science.gov (United States)

    Phansak, Piyaporn; Soonsuwon, Watcharin; Hyten, David L; Song, Qijian; Cregan, Perry B; Graef, George L; Specht, James E

    2016-06-01

    Plant breeders continually generate ever-higher yielding cultivars, but also want to improve seed constituent value, which is mainly protein and oil, in soybean [Glycine max (L.) Merr.]. Identification of genetic loci governing those two traits would facilitate that effort. Though genome-wide association offers one such approach, selective genotyping of multiple biparental populations offers a complementary alternative, and was evaluated here, using 48 F2:3 populations (n = ∼224 plants) created by mating 48 high protein germplasm accessions to cultivars of similar maturity, but with normal seed protein content. All F2:3 progeny were phenotyped for seed protein and oil, but only 22 high and 22 low extreme progeny in each F2:3 phenotypic distribution were genotyped with a 1536-SNP chip (ca 450 bimorphic SNPs detected per mating). A significant quantitative trait locus (QTL) on one or more chromosomes was detected for protein in 35 (73%), and for oil in 25 (52%), of the 48 matings, and these QTL exhibited additive effects of ≥ 4 g kg(-1) and R(2) values of 0.07 or more. These results demonstrated that a multiple-population selective genotyping strategy, when focused on matings between parental phenotype extremes, can be used successfully to identify germplasm accessions possessing large-effect QTL alleles. Such accessions would be of interest to breeders to serve as parental donors of those alleles in cultivar development programs, though 17 of the 48 accessions were not unique in terms of SNP genotype, indicating that diversity among high protein accessions in the germplasm collection is less than what might ordinarily be assumed. Copyright © 2016 Phansak et al.

  8. A new method for finding the minimum free energy pathway of ions and small molecule transportation through protein based on 3D-RISM theory and the string method

    Science.gov (United States)

    Yoshida, Norio

    2018-05-01

    A new method for finding the minimum free energy pathway (MFEP) of ions and small molecule transportation through a protein based on the three-dimensional reference interaction site model (3D-RISM) theory combined with the string method has been proposed. The 3D-RISM theory produces the distribution function, or the potential of mean force (PMF), for transporting substances around the given protein structures. By applying the string method to the PMF surface, one can readily determine the MFEP on the PMF surface. The method has been applied to consider the Na+ conduction pathway of channelrhodopsin as an example.

  9. Dietary Protein and Amino Acid Profiles in Relation to Risk of Dysglycemia: Findings from a Prospective Population-Based Study

    OpenAIRE

    Mirmiran, Parvin; Bahadoran, Zahra; Esfandyari, Saeed; Azizi, Fereidoun

    2017-01-01

    Considering the limited knowledge on the effects of dietary amino acid intake on dysglycemia, we assessed the possible association of dietary protein and amino acid patterns with the risk of pre-diabetes in a prospective population-based study. Participants without diabetes and pre-diabetes (n = 1878) were recruited from the Tehran Lipid and Glucose Study and were followed for a mean of 5.8 years. Their dietary protein and amino acid intakes were assessed at baseline (2006–2008); demographic,...

  10. Extracellular matrix proteins matrix metallopeptidase 9 (MMP9) and soluble intercellular adhesion molecule 1 (sICAM-1) and correlations with clinical staging in euthymic bipolar disorder.

    Science.gov (United States)

    Reininghaus, Eva Z; Lackner, Nina; Birner, Armin; Bengesser, Susanne; Fellendorf, Frederike T; Platzer, Martina; Rieger, Alexandra; Queissner, Robert; Kainzbauer, Nora; Reininghaus, Bernd; McIntyre, Roger S; Mangge, Harald; Zelzer, Sieglinde; Fuchs, Dietmar; Dejonge, Silvia; Müller, Norbert

    2016-03-01

    Matrix metallopeptidase 9 (MMP9) and soluble intercellular adhesion molecule 1 (sICAM-1) are both involved in the restructuring of connective tissues. Evidence also implicates MMP9 and sICAM in cardiovascular and neoplastic diseases, where blood levels may be a marker of disease severity or prognosis. In individuals with bipolar disorder (BD), higher risk for cardiovascular illness has been extensively reported. The aim of this investigation was to measure and compare peripheral levels of serum MMP9 and sICAM in adults with euthymic BD and healthy controls (HC). Furthermore, we focussed on correlations with illness severity and metabolic parameters. MMP9 levels among the BD sample (n = 112) were significantly higher than among the HC (n = 80) (MMP9: F = 9.885, p = 0.002, η(2)  = 0.058) after controlling for confounding factors. Patients with BD in a later, progressive stage of disease showed significantly higher MMP9 as well as sICAM-1 levels compared to patients with BD in an earlier stage of disease (MMP9: F = 5.8, p = 0.018, η(2)  = 0.054; sICAM-1: F = 5.6, p = 0.020, η(2)  = 0.052). Correlation analyses of cognitive measures revealed a negative association between performance on the d2 Test of Attention and MMP9 (r = -0.287, p = 0.018) in the BD sample. Despite the sample being euthymic (i.e., according to conventional criteria) at the time of analysis, we found significant correlations between MMP9 as well as sICAM-1 and subthreshold depressive/hypomanic symptoms. A collection of disparate findings herein point to a role of MMP9 and cICAM-1 in the patho-progressive process of BD: the increased levels of serum MMP9 and sICAM-1, the correlation between higher levels of these parameters, progressive stage, and cognitive dysfunction in BD, and the positive correlation with subthreshold symptoms. As sICAM-1 and MMP9 are reliable biomarkers of inflammatory and early atherosclerotic disease, these markers may provide indications of the

  11. Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

    Science.gov (United States)

    Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

    2016-06-01

    The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Sources of dietary protein in relation to blood pressure in a general Dutch population

    NARCIS (Netherlands)

    Altorf-van der Kuil, W.; Engberink, M.F.; Vedder, M.M.; Boer, J.M.A.; Verschuren, W.M.M.; Geleijnse, J.M.

    2012-01-01

    Background - Little is known about the relation of different dietary protein types with blood pressure (BP). We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20–65 y and not using

  13. Serum, plasma, and dried blood spot high sensitivity C-reactive protein enzyme immunoassay for population research

    OpenAIRE

    Brindle, Eleanor; Fujita, Masako; Shofer, Jane; O’Connor, Kathleen A.

    2010-01-01

    C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus need for a robust high sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to...

  14. Evidence for the Influence of the Iron Regulatory MHC Class I Molecule HFE on Tumor Progression in Experimental Models and Clinical Populations

    Science.gov (United States)

    Weston, Cody; Connor, James

    2014-01-01

    Proteins involved in iron regulation are modifiers of cancer risk and progression. Of these, the HFE protein (high iron gene and its protein product) is of particular interest because of its interaction with both iron handling and immune function and the high rate of genetic polymorphisms resulting in a mutant protein. Clinical studies suggest that HFE polymorphisms increase the risk of certain cancers, but the inconsistent outcomes suggest a more nuanced effect, possibly interacting with other genetic or environmental factors. Some basic science research has been conducted to begin to understand the implications of variant HFE genotype on cancer, but the story is far from complete. In particular, putative mechanisms exist for HFE to affect tumor progression through its role in iron handling and its major histocompatibility complex class I structural features. In this review, the current understanding of the role of HFE in cancer is described and models for future directions are identified. PMID:25520556

  15. Molecule Matters van der Waals Molecules

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 14; Issue 12. Molecule Matters van der Waals Molecules - Noble Gas Clusters are London Molecules! E Arunan. Feature Article Volume 14 Issue 12 December 2009 pp 1210-1222 ...

  16. Genetic diversity and structure analysis based on hordein protein polymorphism in barley landrace populations from jordan

    International Nuclear Information System (INIS)

    Baloch, A.W.; Ali, M.; Baloch, A.M.; Mangan, B.U.N.; Song, W

    2014-01-01

    Jordan is unanimously considered to be one of the centers of genetic diversity for barley, where wild and landraces of barley has been grown under different climatic conditions. The genetic diversity and genetic structure based on hordein polymorphism was assessed in 90 different accessions collected from four different sites of Jordan. A-PAGE was used to reveal hordein polymorphism among the genotypes. A total of 29 distinct bands were identified, out of them 9 bands were distinguished for D, 11 for C, and 9 for the B hordein regions. The observed genetic similarity was an exceptionally high between the populations than expected, which is probably due to high gene flow estimated between them. The genetic diversity parameters were not differ largely among the populations, indicating that local selection of a particular site did not play a key role in shaping genetic diversity. Analysis of molecular variance (AMOVA) revealed significant population structure when accessions were structured according to population site. There was 94% of hordein variation resided within the populations and only 8% present among the populations. Both Bayesian and Principale Coordinate Analysis (PCoA) concordantly demonstrated admixture genotypes of the landraces barley populations. Consequently, none of the population found to be clustered separately according to its population site. It is concluded that this approach can be useful to explore the germplasm for genetic diversity but perhaps is not suitable for determining phylogenic relations in barley. (author)

  17. Growth Patterns in the Irish Pyridoxine Nonresponsive Homocystinuria Population and the Influence of Metabolic Control and Protein Intake.

    Science.gov (United States)

    Purcell, Orla; Coughlan, Aoife; Grant, Tim; McNulty, Jenny; Clark, Anne; Deverell, Deirdre; Mayne, Philip; Hughes, Joanne; Monavari, Ahmad; Knerr, Ina; Crushell, Ellen

    2017-01-01

    A low methionine diet is the mainstay of treatment for pyridoxine nonresponsive homocystinuria (HCU). There are various guidelines for recommended protein intakes for HCU and clinical practice varies. Poor growth has been associated with low cystine levels. This retrospective review of 48 Irish pyridoxine nonresponsive HCU patients assessed weight, height, body mass index (BMI), protein intake, and metabolic control up to 18 years at nine set time points. Patients diagnosed through newborn screening (NBS) were compared to late diagnosed (LD) patients. At 18 years the LD group ( n = 12, mean age at diagnosis 5.09 years) were heavier (estimated effect +4.97 Kg, P = 0.0058) and taller (estimated effect +7.97 cm P = 0.0204) than the NBS group ( n = 36). There was no difference in growth rate between the groups after 10 years of age. The HCU population were heavier and taller than the general population by one standard deviation with no difference in BMI. There was no association between intermittently low cystine levels and height. Three protein intake guidelines were compared; there was no difference in adult height between those who met the lowest of the guidelines (Genetic Metabolic Dietitians International) and those with a higher protein intake.

  18. Population Divergence in Venom Bioactivities of Elapid Snake Pseudonaja textilis: Role of Procoagulant Proteins in Rapid Rodent Prey Incapacitation

    Science.gov (United States)

    Skejić, Jure; Hodgson, Wayne C.

    2013-01-01

    This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD) venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality) and Queensland (Mackay locality) populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver. PMID:23691135

  19. Growth Patterns in the Irish Pyridoxine Nonresponsive Homocystinuria Population and the Influence of Metabolic Control and Protein Intake

    Directory of Open Access Journals (Sweden)

    Orla Purcell

    2017-01-01

    Full Text Available A low methionine diet is the mainstay of treatment for pyridoxine nonresponsive homocystinuria (HCU. There are various guidelines for recommended protein intakes for HCU and clinical practice varies. Poor growth has been associated with low cystine levels. This retrospective review of 48 Irish pyridoxine nonresponsive HCU patients assessed weight, height, body mass index (BMI, protein intake, and metabolic control up to 18 years at nine set time points. Patients diagnosed through newborn screening (NBS were compared to late diagnosed (LD patients. At 18 years the LD group (n=12, mean age at diagnosis 5.09 years were heavier (estimated effect +4.97 Kg, P=0.0058 and taller (estimated effect +7.97 cm P=0.0204 than the NBS group (n=36. There was no difference in growth rate between the groups after 10 years of age. The HCU population were heavier and taller than the general population by one standard deviation with no difference in BMI. There was no association between intermittently low cystine levels and height. Three protein intake guidelines were compared; there was no difference in adult height between those who met the lowest of the guidelines (Genetic Metabolic Dietitians International and those with a higher protein intake.

  20. Population divergence in venom bioactivities of elapid snake Pseudonaja textilis: role of procoagulant proteins in rapid rodent prey incapacitation.

    Directory of Open Access Journals (Sweden)

    Jure Skejić

    Full Text Available This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality and Queensland (Mackay locality populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver.

  1. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro...... conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules...

  2. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, E.; Blicher, T.

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1) correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2) optimal in vitro...... conditions for disulfide bond formation (similar topH 8) and peptide binding (similar topH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct...

  3. Protein structure and phenotypic analysis of pathogenic and population missense variants in STXBP1

    OpenAIRE

    Suri, Mohnish; Evers, Jochem M. G.; Laskowski, Roman A.; O'Brien, Sinead; Baker, Kate; Clayton‐Smith, Jill; Dabir, Tabib; Josifova, Dragana; Joss, Shelagh; Kerr, Bronwyn; Kraus, Alison; McEntagart, Meriel; Morton, Jenny; Smith, Audrey; Splitt, Miranda

    2017-01-01

    Abstract Background Syntaxin‐binding protein 1, encoded by STXBP1, is highly expressed in the brain and involved in fusing synaptic vesicles with the plasma membrane. Studies have shown that pathogenic loss‐of‐function variants in this gene result in various types of epilepsies, mostly beginning early in life. We were interested to model pathogenic missense variants on the protein structure to investigate the mechanism of pathogenicity and genotype–phenotype correlations. Methods We report 11...

  4. Single molecule detection, thermal fluctuation and life

    Science.gov (United States)

    YANAGIDA, Toshio; ISHII, Yoshiharu

    2017-01-01

    Single molecule detection has contributed to our understanding of the unique mechanisms of life. Unlike artificial man-made machines, biological molecular machines integrate thermal noises rather than avoid them. For example, single molecule detection has demonstrated that myosin motors undergo biased Brownian motion for stepwise movement and that single protein molecules spontaneously change their conformation, for switching to interactions with other proteins, in response to thermal fluctuation. Thus, molecular machines have flexibility and efficiency not seen in artificial machines. PMID:28190869

  5. Xylo-Oligosaccharides and Inulin Affect Genotoxicity and Bacterial Populations Differently in a Human Colonic Simulator Challenged with Soy Protein

    Science.gov (United States)

    Christophersen, Claus T.; Petersen, Anne; Licht, Tine R.; Conlon, Michael A.

    2013-01-01

    High dietary intakes of some protein sources, including soy protein, can increase colonic DNA damage in animals, whereas some carbohydrates attenuate this. We investigated whether inulin and xylo-oligosaccharides (XOS) could be protective against DNA strand breaks by adding them to a human colonic simulator consisting of a proximal vessel (PV) (pH 5.5) and a distal vessel (DV) (pH 6.8) inoculated with human faeces and media containing soy protein. Genotoxicity of the liquid phase and microbial population changes in the vessels were measured. Soy protein (3%) was fermented with 1% low amylose cornstarch for 10 day followed by soy protein with 1% XOS or 1% inulin for 10 day. Inulin did not alter genotoxicity but XOS significantly reduced PV genotoxicity and increased DV genotoxicity. Inulin and XOS significantly increased butyrate concentration in the DV but not PV. Numbers of the key butyrate-producing bacterium Faecalibacterium prausnitzii were significantly increased in the PV and DV by inulin but significantly decreased by XOS in both vessels. Other bacteria examined were also significantly impacted by the carbohydrate treatments or by the vessel (i.e., pH). There was a significant overall inverse correlation between levels of damage induced by the ferments and levels of sulphate-reducing bacteria, Bacteroides fragilis, and acetate. In conclusion, dietary XOS can potentially modulate the genotoxicity of the colonic environment and specific bacterial groups and short chain fatty acids may mediate this. PMID:24064573

  6. Interstellar molecules and masers

    International Nuclear Information System (INIS)

    Nguyen-Q-Rieu; Guibert, J.

    1978-01-01

    The study of dense and dark clouds, in which hydrogen is mostly in molecular form, became possible since the discovery of interstellar molecules, emitting in the centimeter and millimeter wavelengths. The molecular lines are generally not in local thermal equilibrium (LTE). Their intensity can often be explained by invoking a population inversion mechanism. Maser emission lines due to OH, H 2 O and SiO molecules are among the most intense molecular lines. The H 2 CO molecule, detected in absorption in front of the cold cosmic background radiation of 2.7 K, illustrates the inverse phenomenon, the antimaser absorption. For a radio transition of frequency v, the inversion rate Δn (relative population difference between the upper and lower level) as well as the maser gain can be determined from the radio observations. In the case of the OH lines in the 2 PIsub(3/2), J=3/2 state, the inversion rates approximately 1 to 2% derived from the observations, are comparable with those obtained in the laboratory. The determination of the excitation mechanisms of the masers, through the statistical equilibrium and radiative transfer equations, implies the knowledge of collisional and radiative transition probabilities. A pumping model, which can satisfactorily explain the radio observations of some interstellar OH clouds, will be discussed [fr

  7. Common variants in the G protein beta3 subunit gene and thyroid disorders in a formerly iodine-deficient population.

    Science.gov (United States)

    Völzke, Henry; Bornhorst, Alexa; Rimmbach, Christian; Petersenn, Holger; Geissler, Ingrid; Nauck, Matthias; Wallaschofski, Henri; Kroemer, Heyo K; Rosskopf, Dieter

    2009-10-01

    Heterotrimeric G proteins are key mediators of signals from membrane receptors-including the thyroid-stimulating hormone (TSH) receptor-to cellular effectors. Gain-of-function mutations in the TSH receptor and the Galpha(S) subunit occur frequently in hyperfunctioning thyroid nodules and differentiated thyroid carcinomas, whereby the T allele of a common polymorphism (825C>T, rs5443) in the G protein beta3 subunit gene (GNB3) is associated with increased G protein-mediated signal transduction and a complex phenotype. The aim of this study was to investigate whether this common polymorphism affects key parameters of thyroid function and morphology and influences the pathogenesis of thyroid diseases in the general population. The population-based cross-sectional Study of Health in Pomerania is a general health survey with focus on thyroid diseases in northeast Germany, a formerly iodine-deficient area. Data from 3428 subjects (1800 men and 1628 women) were analyzed for an association of the GNB3 genotype with TSH, free triiodothyronine and thyroxine levels, urine iodine and thiocyanate excretion, and thyroid ultrasound morphology including thyroid volume, presence of goiter, and thyroid nodules. There was no association between GNB3 genotype status and the functional or morphological thyroid parameters investigated, neither in crude analyses nor upon multivariable analyses including known confounders of thyroid disorders. Based on the data from this large population-based survey, we conclude that the GNB3 825C>T polymorphism does not affect key parameters of thyroid function and morphology in the general population of a formerly iodine-deficient area.

  8. Low-temperature protein dynamics of the B800 molecules in the LH2 light-harvesting complex: spectral hole burning study and comparison with single photosynthetic complex spectroscopy.

    Science.gov (United States)

    Grozdanov, Daniel; Herascu, Nicoleta; Reinot, Tõnu; Jankowiak, Ryszard; Zazubovich, Valter

    2010-03-18

    Previously published and new spectral hole burning (SHB) data on the B800 band of LH2 light-harvesting antenna complex of Rps. acidophila are analyzed in light of recent single photosynthetic complex spectroscopy (SPCS) results (for a review, see Berlin et al. Phys. Life Rev. 2007, 4, 64.). It is demonstrated that, in general, SHB-related phenomena observed for the B800 band are in qualitative agreement with the SPCS data and the protein models involving multiwell multitier protein energy landscapes. Regarding the quantitative agreement, we argue that the single-molecule behavior associated with the fastest spectral diffusion (smallest barrier) tier of the protein energy landscape is inconsistent with the SHB data. The latter discrepancy can be attributed to SPCS probing not only the dynamics of of the protein complex per se, but also that of the surrounding amorphous host and/or of the host-protein interface. It is argued that SHB (once improved models are developed) should also be able to provide the average magnitudes and probability distributions of light-induced spectral shifts and could be used to determine whether SPCS probes a set of protein complexes that are both intact and statistically relevant. SHB results are consistent with the B800 --> B850 energy-transfer models including consideration of the whole B850 density of states.

  9. Plasma surfactant protein D levels and the relation to body mass index in a chinese population

    DEFF Research Database (Denmark)

    Zhao, X M; Wu, Y P; Wei, R

    2007-01-01

    in a Chinese population from the Tai An region comprising 268 individuals. We found that (i) plasma SP-D in the Chinese population was distributed with a median value of 380.2 ng/ml (324.9; 418.7) and a range from 79.4 to 3965.3 ng/ml, (ii) significantly higher plasma SP-D in men than in women...

  10. Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

    Directory of Open Access Journals (Sweden)

    Haddy K S Fye

    Full Text Available Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose

  11. Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

    Directory of Open Access Journals (Sweden)

    Pruksachatkunakorn Chulabhorn

    2006-08-01

    Full Text Available Abstract Background Most group A streptococcal (GAS vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains. Results The emm genes of GAS isolates were sequenced and grouped as 14 different J14-types. The most diversity of J14-types was found in the C1-repeat. The J14.1 type was the major sequence in the C2 and C3-repeats. We have shown that antisera raised against the M protein conserved C-repeat region peptides, J14, J14.1, J14-R1 and J14-R2, commonly found in GAS isolates from the Northern Thai population, are able to kill GAS of multiple different emm types derived from an endemic area. The mean percent of bactericidal activities for all J14 and J14-like peptide antisera against GAS isolates were more than 70%. The mean percent of bactericidal activity was highest for J14 antisera followed by J14-R2, J14.1 and J14-R1 antisera. Conclusion Our study demonstrated that antisera raised against the M protein conserved C-repeat region are able to kill multiple different strains of GAS isolated from the Northern Thai population. Therefore, the four conserved "J14" peptides have the potential to be used as GAS vaccine candidates to prevent streptococcal infections in an endemic area.

  12. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  13. A stochastic algorithm for global optimization and for best populations: A test case of side chains in proteins

    Science.gov (United States)

    Glick, Meir; Rayan, Anwar; Goldblum, Amiram

    2002-01-01

    The problem of global optimization is pivotal in a variety of scientific fields. Here, we present a robust stochastic search method that is able to find the global minimum for a given cost function, as well as, in most cases, any number of best solutions for very large combinatorial “explosive” systems. The algorithm iteratively eliminates variable values that contribute consistently to the highest end of a cost function's spectrum of values for the full system. Values that have not been eliminated are retained for a full, exhaustive search, allowing the creation of an ordered population of best solutions, which includes the global minimum. We demonstrate the ability of the algorithm to explore the conformational space of side chains in eight proteins, with 54 to 263 residues, to reproduce a population of their low energy conformations. The 1,000 lowest energy solutions are identical in the stochastic (with two different seed numbers) and full, exhaustive searches for six of eight proteins. The others retain the lowest 141 and 213 (of 1,000) conformations, depending on the seed number, and the maximal difference between stochastic and exhaustive is only about 0.15 Kcal/mol. The energy gap between the lowest and highest of the 1,000 low-energy conformers in eight proteins is between 0.55 and 3.64 Kcal/mol. This algorithm offers real opportunities for solving problems of high complexity in structural biology and in other fields of science and technology. PMID:11792838

  14. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

    Science.gov (United States)

    Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

  15. Exploring the nature of the H-bonds between the human class II MHC protein, HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide, HA306-318, using the quantum theory of atoms in molecules.

    Science.gov (United States)

    Aray, Yosslen; Aguilera-García, Ricardo; Izquierdo, Daniel R

    2018-01-02

    The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HC aliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) > P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.

  16. Integrating population variation and protein structural analysis to improve clinical interpretation of missense variation: application to the WD40 domain.

    Science.gov (United States)

    Laskowski, Roman A; Tyagi, Nidhi; Johnson, Diana; Joss, Shelagh; Kinning, Esther; McWilliam, Catherine; Splitt, Miranda; Thornton, Janet M; Firth, Helen V; Wright, Caroline F

    2016-03-01

    We present a generic, multidisciplinary approach for improving our understanding of novel missense variants in recently discovered disease genes exhibiting genetic heterogeneity, by combining clinical and population genetics with protein structural analysis. Using six new de novo missense diagnoses in TBL1XR1 from the Deciphering Developmental Disorders study, together with population variation data, we show that the β-propeller structure of the ubiquitous WD40 domain provides a convincing way to discriminate between pathogenic and benign variation. Children with likely pathogenic mutations in this gene have severely delayed language development, often accompanied by intellectual disability, autism, dysmorphology and gastrointestinal problems. Amino acids affected by likely pathogenic missense mutations are either crucial for the stability of the fold, forming part of a highly conserved symmetrically repeating hydrogen-bonded tetrad, or located at the top face of the β-propeller, where 'hotspot' residues affect the binding of β-catenin to the TBLR1 protein. In contrast, those altered by population variation are significantly less likely to be spatially clustered towards the top face or to be at buried or highly conserved residues. This result is useful not only for interpreting benign and pathogenic missense variants in this gene, but also in other WD40 domains, many of which are associated with disease. © The Author 2016. Published by Oxford University Press.

  17. C-reactive protein, insulin resistance and risk of cardiovascular disease: a population-based study

    DEFF Research Database (Denmark)

    Hansen, T.W.; Olsen, M.H.; Rasmussen, S.

    2008-01-01

    BACKGROUND: C-reactive protein (CRP), a marker of inflammation, and insulin resistance (IR), a metabolic disorder, are closely related. CRP and IR have both been identified as significant risk factors of cardiovascular disease (CVD) after adjustment for conventional CVD risk factors...

  18. Changes in microbial populations of WPC34 and WPC80 whey protein during long term storage

    Science.gov (United States)

    The use of whey protein (WPC34 and WPC80) as a food ingredient and as a base for making biodegradable products is increasing. The need to alleviate world hunger in arid and semi-arid regions demands that we investigate the behavior of native bacteria in these products, especially during long term st...

  19. Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

    Directory of Open Access Journals (Sweden)

    Bryan Sands

    Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

  20. Variation of sigma-hole magnitude with M valence electron population in MX(n)Y(4-n) molecules (n = 1-4; M = C, Si, Ge; X, Y = F, Cl, Br).

    Science.gov (United States)

    McDowell, Sean A C; Joseph, Jerelle A

    2014-01-14

    Sigma holes are described as electron-deficient regions on atoms, particularly along the extension of covalent bonds, due to non-uniform electron density distribution on the surface of these atoms. A computational study of MX(n)Y(4-n) molecules (n = 1-4; M = C, Si, Ge; X, Y = F, Cl, Br) was undertaken and it is shown that the relative sigma hole potentials on M due to X-M and Y-M can be adequately explained in terms of the variation in the valence electron population of the central M atom. A model is proposed for the depletion of the M valence electron population which explains the trends in sigma hole strengths, especially those that cannot be accounted for solely on the basis of relative electronegativities.

  1. Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA.

    Science.gov (United States)

    Ren, Hang; Cheyne, Cameron G; Fleming, Aaron M; Burrows, Cynthia J; White, Henry S

    2018-04-18

    Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid-base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current-time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state ( k intra-extra ) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state ( k extra-intra ) remains unchanged. Both k intra-extra and k extra-intra are on the order of 1 × 10 -2 s -1 to 1 × 10 -1 s -1 and remain stable over the time scale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H + into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for k intra-extra ( k H / k D ≈ 5) but a limited KIE for k extra-intra ( k H / k D ≈ 1.3), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries.

  2. Continuous-flow liquid microjunction surface sampling probe connected on-line with high-performance liquid chromatography/mass spectrometry for spatially resolved analysis of small molecules and proteins.

    Science.gov (United States)

    Van Berkel, Gary J; Kertesz, Vilmos

    2013-06-30

    A continuous-flow liquid microjunction surface sampling probe extracts soluble material from surfaces for direct ionization and detection by mass spectrometry. Demonstrated here is the on-line coupling of such a probe with high-performance liquid chromatography/mass spectrometry (HPLC/MS) enabling extraction, separation and detection of small molecules and proteins from surfaces in a spatially resolved (~0.5 mm diameter spots) manner. A continuous-flow liquid microjunction surface sampling probe was connected to a six-port, two-position valve for extract collection and injection to an HPLC column. A QTRAP® 5500 hybrid triple quadrupole linear ion trap equipped with a Turbo V™ ion source operated in positive electrospray ionization (ESI) mode was used for all experiments. The system operation was tested with the extraction, separation and detection of propranolol and associated metabolites from drug dosed tissues, caffeine from a coffee bean, cocaine from paper currency, and proteins from dried sheep blood spots on paper. Confirmed in the tissue were the parent drug and two different hydroxypropranolol glucuronides. The mass spectrometric response for these compounds from different locations in the liver showed an increase with increasing extraction time (5, 20 and 40 s). For on-line separation and detection/identification of extracted proteins from dried sheep blood spots, two major protein peaks dominated the chromatogram and could be correlated with the expected masses for the hemoglobin α and β chains. Spatially resolved sampling, separation, and detection of small molecules and proteins from surfaces can be accomplished using a continuous-flow liquid microjunction surface sampling probe coupled on-line with HPLC/MS detection. Published in 2013. This article is a U.S. Government work and is in the public domain in the USA.

  3. C-reactive protein levels and treatment resistance in schizophrenia - A Danish population-based cohort study

    DEFF Research Database (Denmark)

    Horsdal, Henriette Thisted; Wimberley, Theresa; Benros, Michael Eriksen

    2017-01-01

    -time schizophrenia diagnosis and a baseline C-reactive protein measurement (a commonly available marker of systemic inflammation) from 2000 to 2012. We defined treatment resistance as the earliest observed instance of either clozapine initiation or hospital admission due to schizophrenia after having received......OBJECTIVE: Schizophrenia is associated with increased levels of inflammatory markers. However, it remains unclear whether inflammatory markers are associated with treatment-resistant schizophrenia. METHODS: We conducted a population-based follow-up study among individuals with a first...... (4.0 vs. 3.1 mg/L, p = .13) was observed among the 52 (13.3%) treatment-resistant individuals. Increased levels of C-reactive protein (above 3 mg/L) at baseline were not associated with treatment resistance (adjusted hazard ratio = 0.99, 95% confidence interval [0.56, 1.73]). CONCLUSIONS: C...

  4. Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

    Science.gov (United States)

    Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

    2016-01-01

    Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

  5. An investigation into the population abundance distribution of mRNAs, proteins, and metabolites in biological systems.

    Science.gov (United States)

    Lu, Chuan; King, Ross D

    2009-08-15

    Distribution analysis is one of the most basic forms of statistical analysis. Thanks to improved analytical methods, accurate and extensive quantitative measurements can now be made of the mRNA, protein and metabolite from biological systems. Here, we report a large-scale analysis of the population abundance distributions of the transcriptomes, proteomes and metabolomes from varied biological systems. We compared the observed empirical distributions with a number of distributions: power law, lognormal, loglogistic, loggamma, right Pareto-lognormal (PLN) and double PLN (dPLN). The best-fit for mRNA, protein and metabolite population abundance distributions was found to be the dPLN. This distribution behaves like a lognormal distribution around the centre, and like a power law distribution in the tails. To better understand the cause of this observed distribution, we explored a simple stochastic model based on geometric Brownian motion. The distribution indicates that multiplicative effects are causally dominant in biological systems. We speculate that these effects arise from chemical reactions: the central-limit theorem then explains the central lognormal, and a number of possible mechanisms could explain the long tails: positive feedback, network topology, etc. Many of the components in the central lognormal parts of the empirical distributions are unidentified and/or have unknown function. This indicates that much more biology awaits discovery.

  6. α2-macroglobulin can crosslink multiple Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) molecules and may facilitate adhesion of parasitized erythrocytes

    DEFF Research Database (Denmark)

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J

    2015-01-01

    -macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly....... Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE...

  7. Low-carbohydrate, high-protein score and mortality in a northern Swedish population-based cohort.

    Science.gov (United States)

    Nilsson, L M; Winkvist, A; Eliasson, M; Jansson, J-H; Hallmans, G; Johansson, I; Lindahl, B; Lenner, P; Van Guelpen, B

    2012-06-01

    Long-term effects of carbohydrate-restricted diets are unclear. We examined a low-carbohydrate, high-protein (LCHP) score in relation to mortality. This is a population-based cohort study on adults in the northern Swedish county of Västerbotten. In 37,639 men (1460 deaths) and 39,680 women (923 deaths) from the population-based Västerbotten Intervention Program, deciles of energy-adjusted carbohydrate (descending) and protein (ascending) intake were added to create an LCHP score (2-20 points). Sex-specific hazard ratios (HR) were calculated by Cox regression. Median intakes of carbohydrates, protein and fat in subjects with LCHP scores 2-20 ranged from 61.0% to 38.6%, 11.3% to 19.2% and 26.6% to 41.5% of total energy intake, respectively. High LCHP score (14-20 points) did not predict all-cause mortality compared with low LCHP score (2-8 points), after accounting for saturated fat intake and established risk factors (men: HR for high vs low 1.03 (95% confidence interval (CI) 0.88-1.20), P for continuous = 0.721; women: HR for high vs low 1.10 (95% CI 0.91-1.32), P for continuous = 0.229). For cancer and cardiovascular disease, no clear associations were found. Carbohydrate intake was inversely associated with all-cause mortality, though only statistically significant in women (multivariate HR per decile increase 0.95 (95% CI 0.91-0.99), P = 0.010). Our results do not support a clear, general association between LCHP score and mortality. Studies encompassing a wider range of macronutrient consumption may be necessary to detect such an association.

  8. Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

    Directory of Open Access Journals (Sweden)

    Gil Luiz HS

    2010-11-01

    Full Text Available Abstract Background Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP. The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII, which is the most variable segment of the protein. Methods To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. Results The results suggest that: (i recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

  9. Molecule Matters van der Waals Molecules

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 15; Issue 7. Molecule Matters van der Waals Molecules - Rg•••HF Complexes are Debye Molecules! E Arunan. Feature Article Volume 15 Issue 7 July 2010 pp 667-674. Fulltext. Click here to view fulltext PDF. Permanent link:

  10. Population-based study of high plasma C-reactive protein concentrations among the Inuit of Nunavik.

    Science.gov (United States)

    Labonté, Marie-Eve; Dewailly, Eric; Chateau-Degat, Marie-Ludivine; Couture, Patrick; Lamarche, Benoît

    2012-01-01

    The shift away from traditional lifestyle in the Inuit population over the past few decades has been associated with an increased prevalence of coronary heart disease (CHD) risk factors such as obesity, high blood pressure (BP) and diabetes. However, the impact of this transition on the pro-inflammatory marker high-sensitivity C-reactive protein (hs-CRP) has not been documented. To examine the prevalence of elevated plasma hs-CRP concentrations in Inuit from Nunavik in the province of Quebec (Canada) and identify anthropometric, biochemical and lifestyle risk factors associated with elevated hs-CRP. A population-representative sample of 801 Inuit residents from 14 villages of Nunavik, aged between 18 and 74 years, was included in the analyses. Subjects participated in a clinical session and completed questionnaires on lifestyle. Multivariate logistic regression was used to determine risk factors for elevated hs-CRP. Elevated plasma hs-CRP concentrations (≥ 2 mg/L) were present in 32.7% (95% confidence interval (CI) 29.5-35.8) of the Inuit adult population and were more prevalent among women than among men (36.7% vs. 29.0%, p=0.007). Multivariate logistic regression analysis indicated that every 1 mmHg increase in systolic BP was associated with a 3% increase in the odds of having hs-CRP concentrations ≥ 2 mg/L in the Inuit population (95% CI 1.01-1.04). The combination of older age (≥ 50 vs. Inuit with values that are similar to those seen in Canadian Caucasian populations. Sex, age, waist circumference and systolic BP are major factors that increase the risk of this inflammatory phenotype among Inuit from Nunavik, despite their different lifestyle background compared with Caucasians.

  11. Identification and validation of quantitative trait loci for seed yield, oil and protein contents in two recombinant inbred line populations of soybean.

    Science.gov (United States)

    Wang, Xianzhi; Jiang, Guo-Liang; Green, Marci; Scott, Roy A; Song, Qijian; Hyten, David L; Cregan, Perry B

    2014-10-01

    Soybean seeds contain high levels of oil and protein, and are the important sources of vegetable oil and plant protein for human consumption and livestock feed. Increased seed yield, oil and protein contents are the main objectives of soybean breeding. The objectives of this study were to identify and validate quantitative trait loci (QTLs) associated with seed yield, oil and protein contents in two recombinant inbred line populations, and to evaluate the consistency of QTLs across different environments, studies and genetic backgrounds. Both the mapping population (SD02-4-59 × A02-381100) and validation population (SD02-911 × SD00-1501) were phenotyped for the three traits in multiple environments. Genetic analysis indicated that oil and protein contents showed high heritabilities while yield exhibited a lower heritability in both populations. Based on a linkage map constructed previously with the mapping population and using composite interval mapping and/or interval mapping analysis, 12 QTLs for seed yield, 16 QTLs for oil content and 11 QTLs for protein content were consistently detected in multiple environments and/or the average data over all environments. Of the QTLs detected in the mapping population, five QTLs for seed yield, eight QTLs for oil content and five QTLs for protein content were confirmed in the validation population by single marker analysis in at least one environment and the average data and by ANOVA over all environments. Eight of these validated QTLs were newly identified. Compared with the other studies, seven QTLs for seed yield, eight QTLs for oil content and nine QTLs for protein content further verified the previously reported QTLs. These QTLs will be useful for breeding higher yield and better quality cultivars, and help effectively and efficiently improve yield potential and nutritional quality in soybean.

  12. Effect of storage temperature on survival and recovery of thermal and extrusion injured Escherichia coli populations in whey protein concentrate and corn meal

    Science.gov (United States)

    In a previous study, we reported viability loss of Escherichia coli populations in corn (CP) and whey protein products (WPP) extruded at different temperatures. However, information on the effect of storage temperatures on injured bacterial populations was not addressed. The objective of this study ...

  13. Subretinal Fluid Levels of Signal-Transduction Proteins and Apoptosis Molecules in Macula-Off Retinal Detachment Undergoing Scleral Buckle Surgery.

    Science.gov (United States)

    Carpineto, Paolo; Aharrh-Gnama, Agbeanda; Ciciarelli, Vincenzo; Borrelli, Enrico; Petti, Francesco; Aloia, Raffaella; Lamolinara, Alessia; Di Nicola, Marta; Mastropasqua, Leonardo

    2016-12-01

    To evaluate signal transduction and early apoptosis protein levels in subretinal fluid collected during scleral buckling surgery for macula-off rhegmatogenous retinal detachment (RRD). Our aim was to assess both their relation with RRD features and their influence on the posttreatment outcome. Thirty-three eyes of 33 RRD patients scheduled for scleral buckle surgery were enrolled in the study. Undiluted subretinal fluid samples were collected during surgery and analyzed via magnetic bead-based immunoassay. All patients underwent a complete ophthalmologic evaluation at baseline and at each follow-up visit (months 1, 3, and 6). Moreover, both at baseline and at the postsurgery month 6 visit, the patients were tested by means of spectral-domain optical coherence tomography (SD-OCT) in order to evaluate the average ganglion cell-inner plexiform complex thickness, as well as the photoreceptor inner segment/outer segment junction status. Patients' clinical features (retinal detachment size, detachment duration, and occurrence of proliferative vitreoretinopathy) were associated with several early apoptotic factors (caspase-8, caspase-9, and B-cell lymphoma 2 [Bcl-2]-associated death promoter [BAD]). Furthermore, both early apoptosis factors (caspase-8, Bcl-2, and p53) and signal-transduction proteins (ERK 1/2) were found to influence the postsurgery month 3 OCT characteristics. Signal-transduction proteins and early apoptosis proteins are associated with different clinical features and postsurgery outcomes.

  14. C-reactive protein, insulin resistance and risk of cardiovascular disease: a population-based study

    DEFF Research Database (Denmark)

    Jeppesen, Jørgen; Hansen, Tine Willum; Olsen, Michael H

    2008-01-01

    were recorded at baseline. CRP was determined by a high-sensitivity assay, and IR was determined by the homoeostasis model assessment (HOMA-IR) method. RESULTS: Over a median follow-up of 9.4 years, the incidence of the prespecified CV event, defined as the composite event of CV death, nonfatal...... and HOMA-IR, the hazard ratio (95% confidence interval) of a CV event was 1.33 (1.14-1.55; PHOMA-IR level. CONCLUSION......BACKGROUND: C-reactive protein (CRP), a marker of inflammation, and insulin resistance (IR), a metabolic disorder, are closely related. CRP and IR have both been identified as significant risk factors of cardiovascular disease (CVD) after adjustment for conventional CVD risk factors...

  15. The use of blood protein polymorphism to estimate genetic distance among populations of Indonesian native sheep, St. Croix and Merino

    Directory of Open Access Journals (Sweden)

    Agus Suparyanto

    2002-03-01

    Full Text Available The genetic distance among populations of Indonesia native sheep (Ciamis, Garut, Sumatera and Garahan, St. Croix and Merino were estimated to investigate the genetic relationship among those breeds. Blood protein polymorphism of transferin (Tf, post-transferin (PTf, albumin (Alb, post-albumin (PAlb were detected from blood plasma, while haemoglobine (Hb was detected from erythrocyte using Polyacrylamide Gel Electrophoresis (PAGE. Results of PAGE showed that Tf was controlled by 6 alleles, while Alb by 4 alleles, PTf by 3 Alleles and PAlb and Hb by 2 alleles. Value of breeding coefficient within individual subpopulations (FIS for Tf (-0,0014, Alb (-0,0046 and Hb (0,0256 were not significantly different by noel. These results show that data of gene frequency are still following Hardy-Weinberg Equilibrium and inbreeding inside the sub population did not occur. The closest distance among the native breeds is the subpopulations of Ciamis and Garut due to neighboring area and similar traits of Thin Tail Sheep. The genetic distance of both population to Sumatera Thin Tail Sheep and Garahan Fat Tail are quite far. In addition to that results all Indonesian native breed were distinctly different from St. Croix and Merino.

  16. Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling

    Science.gov (United States)

    Smith, Jessica L.; Stein, David A.; Shum, David; Fischer, Matthew A.; Radu, Constantin; Bhinder, Bhavneet; Djaballah, Hakim; Nelson, Jay A.; Früh, Klaus

    2014-01-01

    ABSTRACT Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds. IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other

  17. Heat shock protein 70 gene polymorphisms are associated with paranoid schizophrenia in the Polish population.

    Science.gov (United States)

    Kowalczyk, Malgorzata; Owczarek, Aleksander; Suchanek, Renata; Paul-Samojedny, Monika; Fila-Danilow, Anna; Borkowska, Paulina; Kucia, Krzysztof; Kowalski, Jan

    2014-03-01

    HSP70 genes have been considered as promising schizophrenia candidate genes based on their protective role in the central nervous system under stress conditions. In this study, we analyzed the potential implication of HSPA1A +190G/C, HSPA1B +1267A/G, and HSPA1L +2437T/C polymorphisms in the susceptibility to paranoid schizophrenia in a homogenous Caucasian Polish population. In addition, we investigated the association of the polymorphisms with the clinical variables of the disease. Two hundred and three patients with paranoid schizophrenia and 243 healthy controls were enrolled in the study. Polymorphisms of HSPA1A, -1B, and -1L genes were genotyped using the PCR-RFLP technique. Analyses were conducted in entire groups and in subgroups that were stratified according to gender. There were significant differences in the genotype and allele frequencies of HSPA1A polymorphism between the patients and controls. The +190CC genotype and +190C allele were over-represented in the patients and significantly increased the risk for developing schizophrenia (OR = 3.45 and OR = 1.61, respectively). Interestingly, such a risk was higher for females with the +190CC genotype than for males with the +190CC genotype (OR = 5.78 vs. OR = 2.76). We also identified the CGT haplotype as a risk haplotype for schizophrenia and demonstrated the effects of HSPA1A and HSPA1B genotypes on the psychopathology and age of onset. Our study provided the first evidence that the HSPA1A polymorphism may potentially increase the risk of developing paranoid schizophrenia. Further independent analyses in different populations to evaluate the role of gender are needed to replicate these results.

  18. [Association of the genetic variations of bone morphogenetic protein 7 gene with diabetes and insulin resistance in Xinjiang Uygur population].

    Science.gov (United States)

    Yan, Zhi-tao; Li, Nan-fang; Guo, Yan-ying; Yao, Xiao-guang; Wang, Hong-mei; Hu, Jun-li

    2011-06-01

    To investigate the association between the genetic variations of the functional region in bone morphogenetic protein gene (BMP7) with type 2 diabetes mellitus in Chinese Uygur individuals. A case-control study was conducted based on epidemiological investigation. A total of 717 Uygur subjects (276 males and 441 females) were selected and divided into two groups: diabetes mellitus group (n = 502, 191 males and 311 females) and control group (n = 215, 85 males and 130 females). All exons, flanking introns and the promoter regions of (BMP7) gene were sequenced in 48 Uygur diabetics. Representative variations were selected according to the minor allele frequency (MAF) and linkage disequilibrium and genotyped using the TaqMan polymerase chain reaction method in 717 Uygur individuals, a relatively isolated general population in a relatively homogeneous environment and a case-control study was conducted to test the association between the genetic variations of (BMP7) gene and type 2 diabetes mellitus. Five novel and 8 known variations in the (BMP7) gene were identified. All genotype distributions were tested for deviations from Hardy-Weinberg equilibrium (P> 0.05). There was significant difference of genotype distribution of rs6025422 between type 2 diabetes mellitus and control groups in the male population (P 0.05), but there was no difference in total and female population (P> 0.05). And the means of fasting blood glucose (FBG), fasting insulin and HOMA-index significantly decreased in individuals with AA, AG and GG genotypes of rs6025422 in male population (Ppopulation (P> 0.05). The logistic regression analysis showed that GG genotype of rs6025422 variation might be a protective factor for diabetes in male (OR= 0.637, 95% confidence interval 0.439-0.923, P< 0.05). The present study suggests that the rs6025422 polymorphism in (BMP7) gene may be associated with diabetes mellitus and insulin resistance in Uygur men.

  19. Anaerobic digestion of cattle offal: protein and lipid-rich substrate degradation and population dynamics of acidogens and methanogens.

    Science.gov (United States)

    Lee, Joonyeob; Koo, Taewoan; Han, Gyuseong; Shin, Seung Gu; Hwang, Seokhwan

    2015-12-01

    Anaerobic digestion of cattle offal was investigated in batch reactors at 35 °C to determine the feasibility of using cattle offal as a feedstock. The organic content [i.e., volatile solids (VS)] of the cattle offal was mainly composed of protein (33.9%) and lipids (46.1%). Hydrolysis along with acidogenesis was monitored to investigate the substrate degradation and generation of intermediate products (e.g., volatile fatty acids, ammonia). Acetate (2.03 g/L), propionate (0.60 g/L), n-butyrate (0.39 g/L), and iso-valerate (0.37 g/L) were major acidogenesis products (91% of total volatile fatty acid concentration). Overall protein and lipid degradation were 82.9 and 81.8%, respectively. Protein degraded first, and four times faster (0.28 day(-1)) than lipid (0.07 day(-1)). Methane yields were 0.52 L CH4/g VSadded and 0.65 L CH4/g VSremoved, indicating that anaerobic digestion of the offal was feasible. A quantitative QPCR assay was conducted to understand the microbial dynamics. The variation patt erns in the gene concentrations successfully indicated the population dynamics of proteolytic and lipolytic acidogens. A fourth-order Runge-Kutta approximation was used to determine the kinetics of the acidogens. The molecular biotechnology approach was appropriate for the evaluation of the acidogenic biokinetics. The maximum growth rate, μ m, halfsaturation coefficients, K s, microbial yield coefficient, Y, cell mass decay rate coefficient, k d, of the proteolytic acidogens were 9.9 day(-1), 37.8 g protein/L, 1.1 × 10(10) copies/g protein, and 3.8 × 10(-1), respectively. Those for the lipolytic acidogens were 1.2 × 10(-1) day(-1), 8.3 g lipid/L, 1.5 × 10(9) copies/g lipid, and 9.9 × 10(-3) day(-1), respectively.

  20. Conserved water molecules in bacterial serine hydroxymethyltransferases.

    Science.gov (United States)

    Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

    2015-10-01

    Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Integrated cancer therapy combined radiotherapy and immunotherapy. The challenge of using Gc protein-derived macrophage activating factor (GcMAF) as a key molecule

    International Nuclear Information System (INIS)

    Uto, Yoshihiro; Hori, Hitoshi

    2013-01-01

    Radiation oncologists know the conflict between radiotherapy and immunotherapy, but now challenged trails of the integrative cancer therapies combined radiation therapy and various immunoreaction/immune therapies begin. We therefore review the recent results of basic research and clinical trial of the integrated cancer therapies which combined radiotherapy and various immune therapies/immunoreaction, and the challenged studies of combined use of radiotherapy and our developed cancer immunotherapy using serum GcMAF which is human serum containing Gc protein-derived macrophage activating factor (GcMAF). (author)

  2. Paternal epigenetic effects of population density on locust phase-related characteristics associated with heat-shock protein expression.

    Science.gov (United States)

    Chen, Bing; Li, Shaoqin; Ren, Qiang; Tong, Xiwen; Zhang, Xia; Kang, Le

    2015-02-01

    Many species exhibit transgenerational plasticity by which environmental cues experienced by either parent can be transmitted to their offspring, resulting in phenotypic variants in offspring to match ancestral environments. However, the manner by which paternal experiences affect offspring plasticity through epigenetic inheritance in animals generally remains unclear. In this study, we examined the transgenerational effects of population density on phase-related traits in the migratory locust Locusta migratoria. Using an experimental design that explicitly controls genetic background, we found that the effects of crowd or isolation rearing on phase plasticity could be inherited to the offspring. The isolation of gregarious locusts resulted in reduced weight in offspring eggs and altered morphometric traits in hatchlings, whereas crowding of solitarious locusts exhibited opposite effects. The consequences of density changes were transmitted by both maternal and paternal inheritance, although the expression of paternal effects was not as pronounced as that of maternal effects. Prominent expression of heat-shock proteins (Hsps), such as Hsp90, Hsp70 and Hsp20.6, could be triggered by density changes. Hsps were significantly upregulated upon crowding but downregulated upon isolation. The variation in parental Hsp expression was also transmitted to the offspring, in which the pattern of inheritance was consistent with that of phase characteristics. These results revealed a paternal effect on phase polyphenism and Hsp expression induced by population density, and defined a model system that could be used to study the paternal epigenetic inheritance of environmental changes. © 2015 John Wiley & Sons Ltd.

  3. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bin; Xu, Bingqian, E-mail: bxu@engr.uga.edu [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); Lou, Zhichao [Single Molecule Study Laboratory, College of Engineering and Nanoscale Science, and Engineering Center, University of Georgia, Athens, Georgia 30605 (United States); College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China); Zhang, Haiqian [College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing 210016 (China)

    2016-03-21

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  4. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    International Nuclear Information System (INIS)

    Wang, Bin; Xu, Bingqian; Lou, Zhichao; Zhang, Haiqian

    2016-01-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  5. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  6. Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

    Science.gov (United States)

    Domingues-Hamdi, Elisa; Vasseur, Corinne; Fournier, Jean-Baptiste; Marden, Michael C; Wajcman, Henri; Baudin-Creuza, Véronique

    2014-01-01

    Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

  7. Dietary Protein and Amino Acid Profiles in Relation to Risk of Dysglycemia: Findings from a Prospective Population-Based Study

    Directory of Open Access Journals (Sweden)

    Parvin Mirmiran

    2017-09-01

    Full Text Available Considering the limited knowledge on the effects of dietary amino acid intake on dysglycemia, we assessed the possible association of dietary protein and amino acid patterns with the risk of pre-diabetes in a prospective population-based study. Participants without diabetes and pre-diabetes (n = 1878 were recruited from the Tehran Lipid and Glucose Study and were followed for a mean of 5.8 years. Their dietary protein and amino acid intakes were assessed at baseline (2006–2008; demographic, lifestyle, and biochemical variables were evaluated at baseline and in follow-up examinations. Pre-diabetes was defined according to the American Diabetes Association criteria. Multivariate Cox proportional hazard regression models, adjusted for potential confounders, were used to estimate the risk of pre-diabetes across tertiles of dietary protein and amino acid pattern scores. The mean age of the participants (44.9% men was 38.3 ± 12.7 years at baseline. Three major amino acid patterns were characterized: (1 higher loads of lysine, methionine, valine, aspartic acids, tyrosine, threonine, isoleucine, leucine, alanine, histidine, and serine; (2 higher loads of glycine, cysteine, arginine, and tryptophan; and (3 higher loads of proline and glutamic acid. Dietary total protein intake Hazard Ratio (HR = 1.13, 95% Confidence Interval (CI = 0.92–1.38 and HR = 1.00, 95% CI = 0.81–1.23, in the second and third tertile, respectively was not related to the development of pre-diabetes. The highest score of second dietary amino acid pattern tended to be associated with a decreased risk of pre-diabetes (HR = 0.81, 95% CI = 0.65–1.01, whereas the third pattern was related to an increased risk in the fully adjusted model (HR = 1.24, 95% CI = 1.02–1.52; p for trend = 0.05. These novel data suggest that the amino acid composition of an individual’s diet may modify their risk of pre-diabetes.

  8. The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

    Science.gov (United States)

    Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

    2018-01-01

    The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

  9. Perspectives of imaging of single protein molecules with the present design of the European XFEL. Pt. 1. X-ray source, beamline optics and instrument simulations

    International Nuclear Information System (INIS)

    Serkez, Svitozar; Kocharyan, Vitali; Saldin, Evgeni; Zagorodnov, Igor; Geloni, Gianluca; Yefanov, Oleksandr

    2014-08-01

    The Single Particles, Clusters and Biomolecules (SPB) instrument at the European XFEL is located behind the SASE1 undulator, and aims to support imaging and structure determination of biological specimen between about 0.1 μm and 1 μm size. The instrument is designed to work at photon energies from 3 keV up to 16 keV. This wide operation range is a cause for challenges to the focusing optics. In particular, a long propagation distance of about 900 m between X-ray source and sample leads to a large lateral photon beam size at the optics. The beam divergence is the most important parameter for the optical system, and is largest for the lowest photon energies and for the shortest pulse duration (corresponding to the lowest charge). Due to the large divergence of nominal X-ray pulses with duration shorter than 10 fs, one suffers diffraction from mirror aperture, leading to a 100-fold decrease in fluence at photon energies around 4 keV, which are ideal for imaging of single biomolecules. The nominal SASE1 output power is about 50 GW. This is very far from the level required for single biomolecule imaging, even assuming perfect beamline and focusing efficiency. Here we demonstrate that the parameters of the accelerator complex and of the SASE1 undulator offer an opportunity to optimize the SPB beamline for single biomolecule imaging with minimal additional costs and time. Start to end simulations from the electron injector at the beginning of the accelerator complex up to the generation of diffraction data indicate that one can achieve diffraction without diffraction with about 0.5 photons per Shannon pixel at near-atomic resolution with 10 13 photons in a 4 fs pulse at 4 keV photon energy and in a 100 nm focus, corresponding to a fluence of 10 23 ph/cm 2 . This result is exemplified using the RNA Pol II molecule as a case study.

  10. Perspectives of imaging of single protein molecules with the present design of the European XFEL. Pt. 1. X-ray source, beamline optics and instrument simulations

    Energy Technology Data Exchange (ETDEWEB)

    Serkez, Svitozar; Kocharyan, Vitali; Saldin, Evgeni; Zagorodnov, Igor [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Geloni, Gianluca [European XFEL GmbH, Hamburg (Germany); Yefanov, Oleksandr [Center for Free-Electron Laser Science, Hamburg (Germany)

    2014-08-15

    The Single Particles, Clusters and Biomolecules (SPB) instrument at the European XFEL is located behind the SASE1 undulator, and aims to support imaging and structure determination of biological specimen between about 0.1 μm and 1 μm size. The instrument is designed to work at photon energies from 3 keV up to 16 keV. This wide operation range is a cause for challenges to the focusing optics. In particular, a long propagation distance of about 900 m between X-ray source and sample leads to a large lateral photon beam size at the optics. The beam divergence is the most important parameter for the optical system, and is largest for the lowest photon energies and for the shortest pulse duration (corresponding to the lowest charge). Due to the large divergence of nominal X-ray pulses with duration shorter than 10 fs, one suffers diffraction from mirror aperture, leading to a 100-fold decrease in fluence at photon energies around 4 keV, which are ideal for imaging of single biomolecules. The nominal SASE1 output power is about 50 GW. This is very far from the level required for single biomolecule imaging, even assuming perfect beamline and focusing efficiency. Here we demonstrate that the parameters of the accelerator complex and of the SASE1 undulator offer an opportunity to optimize the SPB beamline for single biomolecule imaging with minimal additional costs and time. Start to end simulations from the electron injector at the beginning of the accelerator complex up to the generation of diffraction data indicate that one can achieve diffraction without diffraction with about 0.5 photons per Shannon pixel at near-atomic resolution with 10{sup 13} photons in a 4 fs pulse at 4 keV photon energy and in a 100 nm focus, corresponding to a fluence of 10{sup 23}ph/cm{sup 2}. This result is exemplified using the RNA Pol II molecule as a case study.

  11. EVALUATION OF THE ROLE OF INTERLEUKIN-8 (IL -8), SOLUBLE INTERCELLULAR ADHESION MOLECULE-1(SICAM-1) AND EOSINOPHIL CATIONIC PROTEIN (ECP) IN PATHOGENESIS OF BRONCHIAL ASTHMA

    International Nuclear Information System (INIS)

    EL-NASHAR, N.A.; MOSTAFA, A.M.E.; AHMED, S.M.; ABDEL-LATIF, A.

    2008-01-01

    Bronchial asthma remains a leading cause of chronic illness in children. Current theories of the pathogenesis of asthma suggest that airway inflammation is an important determinant of bronchial hyperactivity .The interaction of several inflammatory cells, soluble mediators and adhesion molecules may be important determinants of asthma. Since a better understanding of the underlying mechanisms leading to asthma pathology may yield more specific immunological strategies for the treatment of this disease, this study was designed to investigate the contribution of these markers to airway inflammation. The present study included 25 children with asthma and 15 control children. The asthma cases were 18 males and 7 females ( mean age= 9.36 ± 2.16 years). According to the severity of asthma, patients were classified as mild (n=10), moderate (n=9) and severe (n=6) asthma. They were further classified into allergic asthmatics (extrinsic atopic, n=10) and non-allergic (intrinsic asthmatics, n=15). Estimations of serum levels of IL-8, sICAM-1(by ELISA) and ECP (by flouroimmunoassay) were done. The results of this study revealed that serum levels of IL-8 were significantly higher in asthmatics than in controls. Also, serum levels of it were significantly higher in cases with severe and cases with moderate asthma than in cases with mild asthma. Serum levels of sICAM-1 were significantly higher in asthmatic than in control children, in severe than in moderate, and in both than in mild asthma cases. Levels of ECP were significantly higher in asthmatics than in controls. Also, serum levels of it were related to asthma severity. Furthermore, the three biomarkers showed higher expression in allergic asthmatics versus non- allergies. There were positive correlations of IL-8, sICAM-1, ECP and IgE with each other in asthmatic children that may indicate interaction of these markers in regulation and persistence of inflammatory cascade in asthma through different mechanisms. In

  12. Personalized Therapy Against Preeclampsia by Replenishing Placental Protein 13 (PP13 Targeted to Patients With Impaired PP13 Molecule or Function

    Directory of Open Access Journals (Sweden)

    Hamutal Meiri

    Full Text Available Hypertensive disorders affect about one third of all people aged 20 and above, and are treated with anti-hypertensive drugs. Preeclampsia (PE is one form of such disorders that only develops during pregnancy. It affects ten million pregnant women globally and additionally causes fetal loss and major newborn disabilities. The syndrome's origin is multifactorial, and anti-hypertensive drugs are ineffective in treating it. Biomarkers are helpful for predict its development. Generic drugs, such as low dose aspirin, were proven effective in preventing preterm PE. However, it does not cure the majority of cases and many studies are underway for fighting PE with extended use of additional generic drugs, or through new drug development programs.This review focuses on placental protein 13 (PP13. This protein is only expressed in the placenta. Impaired PP13 DNA structure and/or its reduced mRNA expression leads to lower blood PP13 level that predict a higher risk of developing PE. Two polymorphic PP13 variants have been identified: (1 The promoter PP13 variant with an “A/A” genotype in the -98 position (versus “A/C” or “C/C”. Having the “A/A” genotype is coupled to lower PP13 expression, mainly during placental syncytiotrophoblast differentiation and, if associated with obesity and history of previous preeclampsia, it accurately predicts higher risk for developing the disorder. (2 A thymidine deletion at position 221 causes a frame shift in the open reading frame, and the formation of an early stop codon resulting in the formation of DelT221, a truncated variant of PP13. In pregnant rodents, both short- and long- term replenishment of PP13 causes reversible hypotension and vasodilation of uterine vessels. Long-term exposure is also accompanied by the development of larger placentas and newborns. Also, only w/t PP13 is capable of inducing leukocyte apoptosis, providing maternal immune tolerance to pregnancy.Based on published data, we

  13. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

    Directory of Open Access Journals (Sweden)

    Vikash Verma

    Full Text Available DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

  14. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

    Science.gov (United States)

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

    2015-01-01

    DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

  15. The Association between Total Protein and Vegetable Protein Intake and Low Muscle Mass among the Community-Dwelling Elderly Population in Northern Taiwan

    Directory of Open Access Journals (Sweden)

    Ru-Yi Huang

    2016-06-01

    Full Text Available Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (<13.2% were at a higher risk for low muscle mass (odds ratio (OR 3.03, 95% confidence interval (CI 1.37–6.72 than those with diets in the highest quartile (≥17.2%. Similarly, participants with diets in the lowest quartile of vegetable protein density (<5.8% were at a higher risk for low muscle mass (OR 2.34, 95% CI 1.14–4.83 than those with diets in the highest quartile (≥9.4%. Furthermore, the estimated skeletal muscle mass index increased significantly across the quartiles of total protein density (p = 0.023 and vegetable protein density (p = 0.025. Increasing daily intakes of total protein and vegetable protein densities appears to confer protection against pre-sarcopenia status.

  16. Serum Heat Shock Protein 70 Concentration in Relation to Polycystic Ovary Syndrome in a Non-Obese Chinese Population.

    Directory of Open Access Journals (Sweden)

    Hui Gao

    Full Text Available Polycystic ovary syndrome (PCOS represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70 concentrations and PCOS in a non-obese Chinese population.Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI levels, as well as Insulin resistance index (HOMA-IR (P < 0.05. Identically, markers of oxidative stress (malondialdehyde (MDA, 8-Hydroxy-desoxyguanosine (8-OHdG, Nitric oxide (NO and inflammation (tumor necrosis factor-alpha (TNF-α, C-reactive protein (CRP were markedly increased when compared to controls (P < 0.05. Elevated serum Hsp70 was positively correlated with IR, oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI. The receiver-operating characteristic curve (ROC analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946 vs. 0.822 (95% CI 0.744-0.900; P for difference = 0.015.Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.

  17. Serum Heat Shock Protein 70 Concentration in Relation to Polycystic Ovary Syndrome in a Non-Obese Chinese Population.

    Science.gov (United States)

    Gao, Hui; Meng, Jie; Xu, Mengjing; Zhang, Shun; Ghose, Bishwajit; Liu, Jun; Yao, Ping; Yan, Hong; Wang, Di; Liu, Liegang

    2013-01-01

    Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70) concentrations and PCOS in a non-obese Chinese population. Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI) levels, as well as Insulin resistance index (HOMA-IR) (P oxidative stress (malondialdehyde (MDA), 8-Hydroxy-desoxyguanosine (8-OHdG), Nitric oxide (NO)) and inflammation (tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)) were markedly increased when compared to controls (P oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI). The receiver-operating characteristic curve (ROC) analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946) vs. 0.822 (95% CI 0.744-0.900); P for difference = 0.015). Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.

  18. Analysis of Polymorphisms in the Merozoite Surface Protein-3a Gene and Two Microsatellite Loci in Sri Lankan Plasmodium vivax: Evidence of Population Substructure in Sri Lanka

    DEFF Research Database (Denmark)

    Schousboe, Mette L; Rajakaruna, Rupika S; Amerasinghe, Priyanie H

    2011-01-01

    Abstract. The geographical distribution of genetic variation in Plasmodium vivax samples (N = 386) from nine districts across Sri Lanka is described using three markers; the P. vivax merozoite surface protein-3a (Pvmsp-3a) gene, and the two microsatellites m1501 and m3502. At Pvmsp-3a, 11 alleles....... The results show evidence of high genetic diversity and possible population substructure of P. vivax populations in Sri Lanka....

  19. Population pharmacokinetics and pharmacodynamics of teicoplanin in neonates: making better use of C-reactive protein to deliver individualized therapy.

    Science.gov (United States)

    Ramos-Martín, V; Neely, M N; McGowan, P; Siner, S; Padmore, K; Peak, M; Beresford, M W; Turner, M A; Paulus, S; Hope, W W

    2016-11-01

    There is uncertainty about the optimal teicoplanin regimens for neonates. The study aim was to determine the population pharmacokinetics (PK) of teicoplanin in neonates, evaluate currently recommended regimens and explore the exposure-effect relationships. An open-label PK study was conducted. Neonates from 26 to 44 weeks post-menstrual age were recruited (n = 18). The teicoplanin regimen was a 16 mg/kg loading dose, followed by 8 mg/kg once daily. Therapeutic drug monitoring and dose adjustment were not conducted. A standard two-compartment PK model was developed, followed by models that incorporated weight. A PK/pharmacodynamic (PD) model with C-reactive protein serial measurements as the PD input was fitted to the data. Monte Carlo simulations (n = 5000) were performed using Pmetrics. The AUCs at steady state and the proportion of patients achieving the recommended drug exposures (i.e. C min >15 mg/L) were determined. The study was registered in the European Clinical Trials Database Registry (EudraCT: 2012-005738-12). The PK allometric model best accounted for the observed data. The PK parameters medians were: clearance = 0.435 × (weight/70) 0.75 (L/h); volume = 0.765 (L); K cp  = 1.3 (h -1 ); and K pc  = 0.629 (h -1 ). The individual time-course of C-reactive protein was well described using the Bayesian posterior estimates for each patient. The simulated median AUC 96-120 was 302.3 mg·h/L and the median C min at 120 h was 12.9 mg/L; 38.8% of patients attained a C min >15 mg/L by 120 h. Teicoplanin population PK is highly variable in neonates, weight being the best descriptor of PK variability. A low percentage of neonates were able to achieve C min >15 mg/L. The routine use of therapeutic drug monitoring and improved knowledge on the PD of teicoplanin is required. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e

  20. Socioeconomic and racial/ethnic differentials of C-reactive protein levels: a systematic review of population-based studies

    Directory of Open Access Journals (Sweden)

    Victora Cesar G

    2007-08-01

    Full Text Available Abstract Background Socioeconomic and racial/ethnic factors strongly influence cardiovascular disease outcomes and risk factors. C-reactive protein (CRP, a non-specific marker of inflammation, is associated with cardiovascular risk, and knowledge about its distribution in the population may help direct preventive efforts. A systematic review was undertaken to critically assess CRP levels according to socioeconomic and racial/ethnic factors. Methods Medline was searched through December 2006 for population-based studies examining CRP levels among adults with respect to indicators of socioeconomic position (SEP and/or race/ethnicity. Bibliographies from located studies were scanned and 26 experts in the field were contacted for unpublished work. Results Thirty-two relevant articles were located. Cross-sectional (n = 20 and cohort studies (n = 11 were included, as was the control group of one trial. CRP levels were examined with respect to SEP and race/ethnicity in 25 and 15 analyses, respectively. Of 20 studies that were unadjusted or adjusted for demographic variables, 19 found inverse associations between CRP levels and SEP. Of 15 similar studies, 14 found differences between racial/ethnic groups such that whites had the lowest while blacks, Hispanics and South Asians had the highest CRP levels. Most studies also included adjustment for potential mediating variables in the causal chain between SEP or race/ethnicity and CRP. Most of these studies showed attenuated but still significant associations. Conclusion Increasing poverty and non-white race was associated with elevated CRP levels among adults. Most analyses in the literature are underestimating the true effects of racial/ethnic and socioeconomic factors due to adjustment for mediating factors.

  1. Traffic exposure in a population with high prevalence type 2 diabetes - Do medications influence concentrations of C-reactive protein?

    International Nuclear Information System (INIS)

    Rioux, Christine L.; Tucker, Katherine L.; Brugge, Doug; Gute, David M.; Mwamburi, Mkaya

    2011-01-01

    Type 2 Diabetes (T2D) and particulate air pollution are associated with inflammatory dysregulation. We assessed the modifying effects of diabetes medications on the association of C-reactive protein (CRP), a marker of inflammation, and traffic exposure in adults with T2D (n = 379). CRP concentrations were significantly positively associated with residence ≤100 m of a roadway, >100 m and ≤200 m of a roadway and increased traffic density for individuals using insulin. For individuals using oral hypoglycemic medications (OHAs), CRP was significantly negatively associated with residence >100 m - ≤200 m of a roadway and multiple roadway exposure in an interaction model. Among people with diabetes, individuals on insulin appear to be most vulnerable to the effects of traffic exposure. Disease severity among insulin users may promote the pro-inflammatory response to traffic exposure, though diabetes medications may also modify the response. Possible anti-inflammatory effects of OHAs with traffic exposure merit further evaluation. - Highlights: →We examine traffic exposure in a population with high rates of Type 2 Diabetes. →Differences in CRP were evaluated by traffic levels, medication use and type. →Those on insulin had significantly higher CRP with traffic exposure. →Interaction models demonstrated lower CRP with traffic exposure and OHA use. →Diabetes medications may modify the response to traffic exposure. - Among people with diabetes, individuals on insulin appear to be most vulnerable to the effects of traffic exposure. Diabetes medications may modify the response to traffic.

  2. Traffic exposure in a population with high prevalence type 2 diabetes - Do medications influence concentrations of C-reactive protein?

    Energy Technology Data Exchange (ETDEWEB)

    Rioux, Christine L., E-mail: christine.rioux@tufts.edu [Department of Public Health and Community Medicine, Tufts University, 136 Harrison Avenue, Boston, MA 02111 (United States); Tucker, Katherine L. [Department of Health Science, Northeastern University, Boston, MA (United States); Brugge, Doug [Department of Public Health and Community Medicine, Tufts University, 136 Harrison Avenue, Boston, MA 02111 (United States); Gute, David M. [Department of Civil and Environmental Engineering, Tufts University, Medford, MA (United States); Mwamburi, Mkaya [Department of Public Health and Community Medicine, Tufts University, 136 Harrison Avenue, Boston, MA 02111 (United States)

    2011-08-15

    Type 2 Diabetes (T2D) and particulate air pollution are associated with inflammatory dysregulation. We assessed the modifying effects of diabetes medications on the association of C-reactive protein (CRP), a marker of inflammation, and traffic exposure in adults with T2D (n = 379). CRP concentrations were significantly positively associated with residence {<=}100 m of a roadway, >100 m and {<=}200 m of a roadway and increased traffic density for individuals using insulin. For individuals using oral hypoglycemic medications (OHAs), CRP was significantly negatively associated with residence >100 m - {<=}200 m of a roadway and multiple roadway exposure in an interaction model. Among people with diabetes, individuals on insulin appear to be most vulnerable to the effects of traffic exposure. Disease severity among insulin users may promote the pro-inflammatory response to traffic exposure, though diabetes medications may also modify the response. Possible anti-inflammatory effects of OHAs with traffic exposure merit further evaluation. - Highlights: >We examine traffic exposure in a population with high rates of Type 2 Diabetes. >Differences in CRP were evaluated by traffic levels, medication use and type. >Those on insulin had significantly higher CRP with traffic exposure. >Interaction models demonstrated lower CRP with traffic exposure and OHA use. >Diabetes medications may modify the response to traffic exposure. - Among people with diabetes, individuals on insulin appear to be most vulnerable to the effects of traffic exposure. Diabetes medications may modify the response to traffic.

  3. Adipokines, C-reactive protein and Amyotrophic Lateral Sclerosis - results from a population- based ALS registry in Germany.

    Science.gov (United States)

    Nagel, Gabriele; Peter, Raphael S; Rosenbohm, Angela; Koenig, Wolfgang; Dupuis, Luc; Rothenbacher, Dietrich; Ludolph, Albert C

    2017-06-29

    To investigate the associations of leptin, adiponectin and high-sensitive (hs) C-reactive protein (CRP) with risk and prognosis of amyotrophic lateral sclerosis (ALS). Data from a population-based case-control study in Southern Germany (10/2010-6/2014) of 289 ALS patients (mean age of 65.7 (SD 10.5) years, 59.5% men) and 506 controls were included. During median follow-up of 14.5 months of 279 ALS patients 104 (53.9% men, 68.9 (10.3) years) died. Serum samples were measured for leptin, adiponectin and hs-CRP. Conditional logistic regression was used to estimate ALS risk. Survival models were used to appraise the prognostic value. ALS patients were characterized by lower levels of school education, BMI and smoking prevalence. Adjusted for covariates, leptin was inversely associated with ALS risk (top vs. bottom quartile: OR 0.49; 95% CI 0.29-0.80), while for adiponectin a positive association was found (OR 2.89; 95% CI 1.78-4.68). Among ALS patients increasing leptin concentrations were associated with longer survival (p for trend 0.002), while for adiponectin no association was found (p for trend 0.55). For hs-CRP no association was found. Leptin and adiponectin, two key hormones regulating energy metabolism, were strongly and independently related with ALS risk. Leptin levels were further negatively related with overall survival of ALS patients.

  4. The Molecule Cloud - compact visualization of large collections of molecules

    Directory of Open Access Journals (Sweden)

    Ertl Peter

    2012-07-01

    Full Text Available Abstract Background Analysis and visualization of large collections of molecules is one of the most frequent challenges cheminformatics experts in pharmaceutical industry are facing. Various sophisticated methods are available to perform this task, including clustering, dimensionality reduction or scaffold frequency analysis. In any case, however, viewing and analyzing large tables with molecular structures is necessary. We present a new visualization technique, providing basic information about the composition of molecular data sets at a single glance. Summary A method is presented here allowing visual representation of the most common structural features of chemical databases in a form of a cloud diagram. The frequency of molecules containing particular substructure is indicated by the size of respective structural image. The method is useful to quickly perceive the most prominent structural features present in the data set. This approach was inspired by popular word cloud diagrams that are used to visualize textual information in a compact form. Therefore we call this approach “Molecule Cloud”. The method also supports visualization of additional information, for example biological activity of molecules containing this scaffold or the protein target class typical for particular scaffolds, by color coding. Detailed description of the algorithm is provided, allowing easy implementation of the method by any cheminformatics toolkit. The layout algorithm is available as open source Java code. Conclusions Visualization of large molecular data sets using the Molecule Cloud approach allows scientists to get information about the composition of molecular databases and their most frequent structural features easily. The method may be used in the areas where analysis of large molecular collections is needed, for example processing of high throughput screening results, virtual screening or compound purchasing. Several example visualizations of large

  5. Single-molecule dynamics in nanofabricated traps

    Science.gov (United States)

    Cohen, Adam

    2009-03-01

    The Anti-Brownian Electrokinetic trap (ABEL trap) provides a means to immobilize a single fluorescent molecule in solution, without surface attachment chemistry. The ABEL trap works by tracking the Brownian motion of a single molecule, and applying feedback electric fields to induce an electrokinetic motion that approximately cancels the Brownian motion. We present a new design for the ABEL trap that allows smaller molecules to be trapped and more information to be extracted from the dynamics of a single molecule than was previously possible. In particular, we present strategies for extracting dynamically fluctuating mobilities and diffusion coefficients, as a means to probe dynamic changes in molecular charge and shape. If one trapped molecule is good, many trapped molecules are better. An array of single molecules in solution, each immobilized without surface attachment chemistry, provides an ideal test-bed for single-molecule analyses of intramolecular dynamics and intermolecular interactions. We present a technology for creating such an array, using a fused silica plate with nanofabricated dimples and a removable cover for sealing single molecules within the dimples. With this device one can watch the shape fluctuations of single molecules of DNA or study cooperative interactions in weakly associating protein complexes.

  6. C-reactive protein implications in new-onset hypertension in a healthy population initially aged 65 years : the Proof study

    NARCIS (Netherlands)

    Dauphinot, Virginie; Roche, Frederic; Kossovsky, Michel P.; Schott, Anne-Marie; Pichot, Vincent; Gaspoz, Jean-Michel; Gosse, Philippe; Barthelemy, Jean-Claude

    Background Because inflammation is known to be related with several cardiovascular diseases, we sought to determine whether C-reactive protein (CRP) might precede the onset of hypertension. Methods The study population was selected from the Proof study cohort including 1011 individuals initially

  7. Associations of Protein-Energy Wasting Syndrome Criteria With Body Composition and Mortality in the General and Moderate Chronic Kidney Disease Populations in the United States.

    Science.gov (United States)

    Beddhu, Srinivasan; Chen, Xiaorui; Wei, Guo; Raj, Dominic; Raphael, Kalani L; Boucher, Robert; Chonchol, Michel B; Murtaugh, Maureen A; Greene, Tom

    2017-05-01

    It is unknown whether the criteria used to define Protein-energy wasting (PEW) syndrome in dialysis patients reflect protein or energy wasting in the general and moderate CKD populations. In 11,834 participants in the 1999-2004 National Health and Nutrition Examination Survey, individual PEW syndrome criteria and the number of PEW syndrome categories were related to lean body and fat masses (measured by dual-energy absorptiometry) using linear regression in the entire cohort and CKD sub-population. Serum chemistry, body mass and muscle mass PEW criteria tended to be associated with lower lean body and fat masses, but the low dietary protein and energy intake criteria were associated with significantly higher protein and energy stores. When the number of PEW syndrome categories was defined by non-dietary categories alone, there was a monotonic inverse relationship with lean body and fat masses and strong positive relationship with mortality. In contrast, when dietary category alone was present, mean BMI was in the obesity range; additional presence of two non-dietary categories was associated with lower BMI and lower lean body and fat masses. Thus, the association of dietary category plus two additional non-dietary categories with lower protein or energy stores was driven by the presence of the two non-dietary categories. Results were similar in CKD subgroup. Hence, a definition of PEW syndrome without dietary variables has face validity and reflects protein or energy wasting.

  8. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  9. Kidney injury molecule-1 and microalbuminuria levels in Zambian ...

    African Journals Online (AJOL)

    Kidney injury molecule-1 and microalbuminuria levels in Zambian population: biomarkers of kidney injury. Mildred Zulu, Trevor Kaile, Timothy Kantenga, Chisanga Chileshe, Panji Nkhoma, Musalula Sinkala ...

  10. Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Harris, Nicholas; Koppel, Juraj; Zsila, Ferenc; Juhas, Stefan; Il'kova, Gabriela; Kogan, Faina Yurgenzon; Lahmy, Orly; Wildbaum, Gizi; Karin, Nathan; Zhuk, Regina; Gregor, Paul

    2016-04-01

    Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

  11. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  12. Formation of Ultracold Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Cote, Robin [Univ. of Connecticut, Storrs, CT (United States)

    2016-01-28

    Advances in our ability to slow down and cool atoms and molecules to ultracold temperatures have paved the way to a revolution in basic research on molecules. Ultracold molecules are sensitive of very weak interactions, even when separated by large distances, which allow studies of the effect of those interactions on the behavior of molecules. In this program, we have explored ways to form ultracold molecules starting from pairs of atoms that have already reached the ultracold regime. We devised methods that enhance the efficiency of ultracold molecule production, for example by tuning external magnetic fields and using appropriate laser excitations. We also investigates the properties of those ultracold molecules, especially their de-excitation into stable molecules. We studied the possibility of creating new classes of ultra-long range molecules, named macrodimers, thousand times more extended than regular molecules. Again, such objects are possible because ultra low temperatures prevent their breakup by collision. Finally, we carried out calculations on how chemical reactions are affected and modified at ultracold temperatures. Normally, reactions become less effective as the temperature decreases, but at ultracold temperatures, they can become very effective. We studied this counter-intuitive behavior for benchmark chemical reactions involving molecular hydrogen.

  13. Reduced high-molecular-weight adiponectin and elevated high-sensitivity C-reactive protein are synergistic risk factors for metabolic syndrome in a large-scale middle-aged to elderly population: the Shimanami Health Promoting Program Study.

    Science.gov (United States)

    Tabara, Yasuharu; Osawa, Haruhiko; Kawamoto, Ryuichi; Tachibana-Iimori, Rieko; Yamamoto, Miyuki; Nakura, Jun; Miki, Tetsuro; Makino, Hideich; Kohara, Katsuhiko

    2008-03-01

    In Western countries, one of the most important modifiable targets for the prevention of cardiovascular diseases is metabolic syndrome. Adiponectin is an adipose tissue-specific plasma protein that inversely associates with metabolic syndrome. Among several molecular isoforms, high-molecular-weight (HMW) complex is considered the active form. Increased serum high-sensitivity C-reactive protein (hsCRP) concentration also associates with metabolic syndrome, and adiponectin could modulate plasma C-reactive protein levels. Here, through cross-sectional investigation, we investigated whether reduced HMW adiponectin and increased hsCRP levels in plasma are synergistically associated with metabolic syndrome. Measurement of HMW complex of adiponectin is one of the novelties of this study. We analyzed 1845 community-dwelling middle-aged to elderly subjects (62+/-13 yr). Plasma HMW adiponectin levels were measured by ELISA. Clinical parameters were obtained from the subjects' personal health records, evaluated at their annual medical check-up. Each component of metabolic syndrome, except for raised blood pressure, showed significantly lower plasma HMW adiponectin concentrations for both men and women (P<0.001). In contrast, plasma hsCRP levels were significantly higher in subjects with metabolic disorders (P<0.001). After adjusting for other confounding factors, HMW adiponectin [log normalized, odds ratio 0.084 (95% confidence interval 0.050-0.142), P<0.001] and hsCRP [3.009 (2.175-4.163), P<0.001] were identified as independent determinants of metabolic syndrome. In addition to the direct associations, we also observed a synergistic effect between these two molecules (F=11.8, P<0.001). Reduced HMW adiponectin and elevated hsCRP are synergistically associated with the accumulation of metabolic disorders. The combination of these markers would be useful for identifying at-risk populations.

  14. Effect of inclusion of different levels of silage on rumen microbial population and microbial protein synthesis in dairy steers fed on rice straw

    Directory of Open Access Journals (Sweden)

    Thien Truong Giang Nguyen

    2017-02-01

    Full Text Available Objective Leucaena leucocephala (Leucaena is a perennial tropical legume that can be directly grazed or harvested and offered to ruminants as hay, silage, or fresh. However, Leucaena contain phenolic compounds, which are considered anti-nutritional factors as these may reduce intake, digestibility and thus animal performance. Therefore, the objective of this experiment was to determine effects of Leucaena silage (LS feeding levels on rumen microbial populations, N-balance and microbial protein synthesis in dairy steers. Methods Four, rumen fistulated dairy steers with initial weight of 167±12 kg were randomly assigned to receive dietary treatments according to a 4×4 Latin square design. Treatments were as followings: T1 = untreated rice straw (RS; Control, T2 = 70% RS+30% LS, T3 = 40% RS+60% LS, and T4 = 100% LS. Dairy steers were fed rice straw and LS ad libitum and supplemented with concentrate at 0.2% of body weight/d. Results Results revealed that the rumen microbial population, especially cellulolytic, proteolytic bacteria and fungal zoospores were enhanced in steers that received 60% of LS (p0.05. Protozoal population was linearly decreased with increasing level of LS (p<0.05. Moreover, N-balance and microbial protein synthesis were enhanced by LS feeding (p<0.05 and were the highest in 60% LS group. Conclusion Based on this study, it could be concluded that replacement of RS with 60% LS significantly improved microbial population and microbial protein synthesis in diary steers.

  15. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    along the reaction coordinate. The results call for a modified view of the mechanical response of native states which may be relevant in an evolutionary sense for functionality and for biomolecular design. A vastly more complex folding behavior was observed for the two-domain neuronal calcium sensor 1...

  16. Proteins Are the Body's Worker Molecules

    Science.gov (United States)

    ... remains one of the great challenges in structural biology. Although researchers have teased out some general rules ... the launch, in the 1990s, of genome sequencing projects. These ongoing projects give scientists ready access to ...

  17. Organic Molecules in Meteorites

    Science.gov (United States)

    Martins, Zita

    2015-08-01

    Carbonaceous meteorites are primitive samples from the asteroid belt, containing 3-5wt% organic carbon. The exogenous delivery of organic matter by carbonaceous meteorites may have contributed to the organic inventory of the early Earth. The majority (>70%) of the meteoritic organic material consist of insoluble organic matter (IOM) [1]. The remaining meteoritic organic material (meteorites contain soluble organic molecules with different abundances and distributions, which may reflect the extension of aqueous alteration or thermal metamorphism on the meteorite parent bodies. Extensive aqueous alteration on the meteorite parent body may result on 1) the decomposition of α-amino acids [5, 6]; 2) synthesis of β- and γ-amino acids [2, 6-9]; 3) higher relative abundances of alkylated polycyclic aromatic hydrocarbons (PAHs) [6, 10]; and 4) higher L-enantiomer excess (Lee) value of isovaline [6, 11, 12].The soluble organic content of carbonaceous meteorites may also have a contribution from Fischer-Tropsch/Haber-Bosch type gas-grain reactions after the meteorite parent body cooled to lower temperatures [13, 14].The analysis of the abundances and distribution of the organic molecules present in meteorites helps to determine the physical and chemical conditions of the early solar system, and the prebiotic organic compounds available on the early Earth.[1] Cody and Alexander (2005) GCA 69, 1085. [2] Cronin and Chang (1993) in: The Chemistry of Life’s Origin. pp. 209-258. [3] Martins and Sephton (2009) in: Amino acids, peptides and proteins in organic chemistry. pp. 1-42. [4] Martins (2011) Elements 7, 35. [5] Botta et al. (2007) MAPS 42, 81. [6] Martins et al. (2015) MAPS, in press. [7] Cooper and Cronin (1995) GCA 59, 1003. [8] Glavin et al. (2006) MAPS. 41, 889. [9] Glavin et al. (2011) MAPS 45, 1948. [10] Elsila et al. (2005) GCA 5, 1349. [11] Glavin and Dworkin (2009) PNAS 106, 5487. [12] Pizzarello et al. (2003) GCA 67, 1589. [13] Chan et al. (2012) MAPS. 47, 1502

  18. Extracting Models in Single Molecule Experiments

    Science.gov (United States)

    Presse, Steve

    2013-03-01

    Single molecule experiments can now monitor the journey of a protein from its assembly near a ribosome to its proteolytic demise. Ideally all single molecule data should be self-explanatory. However data originating from single molecule experiments is particularly challenging to interpret on account of fluctuations and noise at such small scales. Realistically, basic understanding comes from models carefully extracted from the noisy data. Statistical mechanics, and maximum entropy in particular, provide a powerful framework for accomplishing this task in a principled fashion. Here I will discuss our work in extracting conformational memory from single molecule force spectroscopy experiments on large biomolecules. One clear advantage of this method is that we let the data tend towards the correct model, we do not fit the data. I will show that the dynamical model of the single molecule dynamics which emerges from this analysis is often more textured and complex than could otherwise come from fitting the data to a pre-conceived model.

  19. The status of molecules

    International Nuclear Information System (INIS)

    Barnes, T.; Oak Ridge National Lab., TN; Tennessee Univ., Knoxville, TN

    1994-06-01

    This report summarizes the experimental and theoretical status of hadronic molecules, which are weakly-bound states of two or more hadrons. We begin with a brief history of the subject and discuss a few good candidates, and then abstract some signatures for molecules which may be of interest in the classification of possible molecule states. Next we argue that a more general understanding of 2 → 2 hadron-hadron scattering amplitudes will be crucial for molecule searches, and discuss some of our recent work in this area. We conclude with a discussion of a few more recent molecule candidates (notably the f o (1710)) which are not well established as molecules but satisfy some of the expected signatures. (Author)

  20. Cold Rydberg molecules

    Science.gov (United States)

    Raithel, Georg; Zhao, Jianming

    2017-04-01

    Cold atomic systems have opened new frontiers at the interface of atomic and molecular physics. These include research on novel types of Rydberg molecules. Three types of molecules will be reviewed. Long-range, homonuclear Rydberg molecules, first predicted in [1] and observed in [2], are formed via low-energy electron scattering of the Rydberg electron from a ground-state atom within the Rydberg atom's volume. The binding mostly arises from S- and P-wave triplet scattering. We use a Fermi model that includes S-wave and P-wave singlet and triplet scattering, the fine structure coupling of the Rydberg atom and the hyperfine structure coupling of the 5S1/2 atom (in rubidium [3]). The hyperfine structure gives rise to mixed singlet-triplet potentials for both low-L and high-L Rydberg molecules [3]. A classification into Hund's cases [3, 4, 5] will be discussed. The talk further includes results on adiabatic potentials and adiabatic states of Rydberg-Rydberg molecules in Rb and Cs. These molecules, which have even larger bonding length than Rydberg-ground molecules, are formed via electrostatic multipole interactions. The leading interaction term of neutral Rydberg-Rydberg molecules is between two dipoles, while for ionic Rydberg molecules it is between a dipole and a monopole. NSF (PHY-1506093), NNSF of China (61475123).

  1. Polymorphism in SFTPD gene affects assembly and constitutional serum levels of surfactant protein D in a Lebanese population

    DEFF Research Database (Denmark)

    Fakih, Dalia; Chamat, Soulaima; Medlej-Hashim, Myrna

    2014-01-01

    Surfactant protein D (SP-D), an oligomeric lung-derived lectin, has essential roles in innate immunity. It can be measured in serum. Previous studies have shown that constitutional SP-D serum levels and the protein degree of multimerization are genetically influenced. We aimed to establish the di...

  2. Association of Animal and Plant Proteins Intake with Hypertension in Iranian Adult Population: Isfahan Healthy Heart Program

    Directory of Open Access Journals (Sweden)

    Sanaz Mehrabani

    2017-01-01

    Full Text Available Background: There is evidence regarding the relationship between dietary proteins intake and blood pressure (BP, but they had inconsistent results. Therefore, this study was designed to assess the association between different kinds of protein intake (animal and plant protein and BP. Materials and Methods: Data were collected from Isfahan Healthy Heart Program. We performed a cross-sectional study among 9660 randomly selected Iranian adults aged ≥19-year-old that they were selected from three large Iranian regions in 2007. A simplified validated 48-item-food frequency questionnaire was used to assess dietary intake including all kinds of protein. Systolic and diastolic BPs were measured in duplicate by trained personnel using a standard protocol. Multivariable regressions were applied to assess the relationship between protein intake and BP levels and the presence of hypertension (HTN. Results: More frequent consumption of animal, plant, and total protein intake were inversely associated with BP in a crude model (P < 0.001; however, after adjustment for potential confounders this relationship remained only for plant protein (P = 0.04. The risk of HTN occurrence decreased in the highest quintile of total and plant protein consumption by 19% (odds ratio [OR] = 0.81; confidence interval [CI]: [0.65–0.96]; P for trend = 0.004 and 18% (OR = 0.82; [CI: (0.67–0.94]; P for trend = 0.03, respectively. Conclusions: More frequent protein intake, especially plant protein consumption was inversely associated with BP and risk of HTN among Iranian adults.

  3. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  4. Molecule of the Month

    Indian Academy of Sciences (India)

    Atoms in a molecule generally prefer, particularly among the neighbouring ones, certain optimmn geometrical relationships. These are manifested in specific ranges of bond lengths, bond angles, torsion angles etc. As it always happens, chemists are interested in making molecules where these 'standard relationships' are ...

  5. Molecule of the Month

    Indian Academy of Sciences (India)

    Cyclo bu tadiene (1) has been one of the most popular molecules for experimentalists and theoreticians. This molecule is unstable as . it is antiaromatic ( 4,n electrons in a cyclic array). Even though some highly substituted cyclobutadienes, for example, compound 2 and the Fe(CO)3 complex of cyclobutadiene (3) are ...

  6. Single-Molecule Spectroscopy

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 2. Single-Molecule Spectroscopy: Every Molecule is Different! Kankan Bhattacharyya. General Article Volume 20 Issue 2 February 2015 pp 151-164. Fulltext. Click here to view fulltext PDF. Permanent link: