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Sample records for surface protein level

  1. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2012-01-01

    Full Text Available This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM. Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables.

  2. Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

    Science.gov (United States)

    Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

    2014-01-01

    A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions

  3. Low level chemiluminescence measurement of the binding of 8-methoxypsoralen to proteins and lymphocytic surfaces

    International Nuclear Information System (INIS)

    Lange, B.

    1980-01-01

    Photochemotherapy with 8-methoxypsoralen (8-MOP) and longwave ultraviolet light is beneficial in such different disorders like psoriasis, lichen planus, and mykosis fungoides. In contrast to a widely accepted hypothesis 8-MOP does not solely bind to nucleic acid, but also to certain proteins. The mechanism of this binding as well as the precise binding area are unknown. Therefore the UV-provoked reactions of 8-MOP with a lipid mixture, a glucosaminoglycan solution, a protein solution, and lymphocyte suspensions, respectively were investigated using low level chemiluminescence (LLCL). It was found an 8-MOP concentration-dependent decrease of LLCL intensity in the lymphocyte suspensions (10 3 to 10 4 cells/μl). This effect is result of the diminution of the photoactive 8-MOP content of the solution. 8-MOP binds quickly and in the course of a free radical reaction to lymphocytic surfaces and coincidentally loses its potency to start LLCL-detectable free radical chain responses. (author)

  4. Tumor cell surface proteins

    International Nuclear Information System (INIS)

    Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

    1982-01-01

    Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

  5. 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

  6. Characterizing the statistical properties of protein surfaces

    Science.gov (United States)

    Bak, Ji Hyun; Bitbol, Anne-Florence; Bialek, William

    Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of the shapes of their surfaces. We take into account the high-resolution structures of E.coli non-DNA-binding cytoplasmic proteins, retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the protein surfaces at two levels: First, we study the intrinsic dimensionality at the level of the ensemble of the surface objects. Second, at the level of the individual surfaces, we determine the scale of shape variation. We further discuss how the dimensionality of the shape space is linked to the statistical properties of individual protein surfaces. Jhb and WB acknowledge support from National Science Foundation Grants PHY-1305525 and PHY-1521553. AFB acknowledges support from the Human Frontier Science Program.

  7. Hydrophobic patches on protein surfaces

    NARCIS (Netherlands)

    Lijnzaad, P.

    2007-01-01

    Hydrophobicity is a prime determinant of the structure and function of proteins. It is the driving force behind the folding of soluble proteins, and when exposed on the surface, it is frequently involved in recognition and binding of ligands and other proteins. The energetic cost of

  8. Surface expression and subunit specific control of steady protein levels by the Kv7.2 helix A-B linker.

    Directory of Open Access Journals (Sweden)

    Paloma Aivar

    Full Text Available Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.

  9. Low level of sequence diversity at merozoite surface protein-1 locus of Plasmodium ovale curtisi and P. ovale wallikeri from Thai isolates.

    Science.gov (United States)

    Putaporntip, Chaturong; Hughes, Austin L; Jongwutiwes, Somchai

    2013-01-01

    The merozoite surface protein-1 (MSP-1) is a candidate target for the development of blood stage vaccines against malaria. Polymorphism in MSP-1 can be useful as a genetic marker for strain differentiation in malarial parasites. Although sequence diversity in the MSP-1 locus has been extensively analyzed in field isolates of Plasmodium falciparum and P. vivax, the extent of variation in its homologues in P. ovale curtisi and P. ovale wallikeri, remains unknown. Analysis of the mitochondrial cytochrome b sequences of 10 P. ovale isolates from symptomatic malaria patients from diverse endemic areas of Thailand revealed co-existence of P. ovale curtisi (n = 5) and P. ovale wallikeri (n = 5). Direct sequencing of the PCR-amplified products encompassing the entire coding region of MSP-1 of P. ovale curtisi (PocMSP-1) and P. ovale wallikeri (PowMSP-1) has identified 3 imperfect repeated segments in the former and one in the latter. Most amino acid differences between these proteins were located in the interspecies variable domains of malarial MSP-1. Synonymous nucleotide diversity (πS) exceeded nonsynonymous nucleotide diversity (πN) for both PocMSP-1 and PowMSP-1, albeit at a non-significant level. However, when MSP-1 of both these species was considered together, πS was significantly greater than πN (pdiversity at this locus prior to speciation. Phylogenetic analysis based on conserved domains has placed PocMSP-1 and PowMSP-1 in a distinct bifurcating branch that probably diverged from each other around 4.5 million years ago. The MSP-1 sequences support that P. ovale curtisi and P. ovale wallikeri are distinct species. Both species are sympatric in Thailand. The low level of sequence diversity in PocMSP-1 and PowMSP-1 among Thai isolates could stem from persistent low prevalence of these species, limiting the chance of outcrossing at this locus.

  10. Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria

    DEFF Research Database (Denmark)

    Dodoo, D; Theander, T G; Kurtzhals, J A

    1999-01-01

    malaria season in April and after the season in November. Using enzyme-linked immunosorbent assay, we measured antibody responses to recombinant gluthathione S-transferase-PfMSP119 fusion proteins corresponding to the Wellcome and MAD20 allelic variants in these samples. Prevalence of antibodies......The 19-kDa conserved C-terminal part of the Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is a malaria vaccine candidate antigen, and human antibody responses to PfMSP119 have been associated with protection against clinical malaria. In this longitudinal study carried out in an area...

  11. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  12. Rapid comparison of properties on protein surface.

    Science.gov (United States)

    Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

    2008-10-01

    The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure.

  13. RPE cell surface proteins in normal and dystrophic rats

    International Nuclear Information System (INIS)

    Clark, V.M.; Hall, M.O.

    1986-01-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

  14. 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

  15. Interactions between whey proteins and kaolinite surfaces

    International Nuclear Information System (INIS)

    Barral, S.; Villa-Garcia, M.A.; Rendueles, M.; Diaz, M.

    2008-01-01

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered

  16. Interactions between whey proteins and kaolinite surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Barral, S. [Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Villa-Garcia, M.A. [Department of Organic and Inorganic Chemistry, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)], E-mail: mavg@uniovi.es; Rendueles, M. [Project Management Area, University of Oviedo, Independencia 13, 33004 Oviedo (Spain); Diaz, M. [Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)

    2008-07-15

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered.

  17. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.

    1989-01-01

    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  18. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    International Nuclear Information System (INIS)

    Dooley, J.S.G.; Trust, T.J.

    1988-01-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125 I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to 125 I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein

  19. Functional dynamics of cell surface membrane proteins.

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Proteins in solution: Fractal surfaces in solutions

    Directory of Open Access Journals (Sweden)

    R. Tscheliessnig

    2016-02-01

    Full Text Available The concept of the surface of a protein in solution, as well of the interface between protein and 'bulk solution', is introduced. The experimental technique of small angle X-ray and neutron scattering is introduced and described briefly. Molecular dynamics simulation, as an appropriate computational tool for studying the hydration shell of proteins, is also discussed. The concept of protein surfaces with fractal dimensions is elaborated. We finish by exposing an experimental (using small angle X-ray scattering and a computer simulation case study, which are meant as demonstrations of the possibilities we have at hand for investigating the delicate interfaces that connect (and divide protein molecules and the neighboring electrolyte solution.

  1. Radiation Level Changes at RAM Package Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Opperman, Erich [Washington Savannah River Company; Hawk, Mark B [ORNL; Kapoor, Ashok [U.S. Department of Energy, Office of Packaging and Transportation; Natali, Ronald [R. B. Natali Consulting, Inc.

    2010-01-01

    This paper will explore design considerations required to meet the regulations that limit radiation level variations at external surfaces of radioactive material (RAM) packages. The radiation level requirements at package surfaces (e.g. TS-R-1 paragraphs 531 and 646) invoke not only maximum radiation levels, but also strict limits on the allowable increase in the radiation level during transport. This paper will explore the regulatory requirements by quantifying the amount of near surface movement and/or payload shifting that results in a 20% increase in the radiation level at the package surface. Typical IP-2, IP-3, Type A and Type B packaging and source geometries will be illustrated. Variations in surface radiation levels are typically the result of changes in the geometry of the surface due to an impact, puncture or crush event, or shifting and settling of radioactive contents.

  2. Quantitation of secretory protein levels by radioimmunoassay

    International Nuclear Information System (INIS)

    Klein, J.L.; Dawson, J.R.

    1978-01-01

    A radioimmunoassay was designed for the detection of secretory protein, a component of secretory immunoglobulin A, in human serum. The assay uses free secretory protein isolated from human colostrum, and antisera raised in rabbits to be purified antigen. The mean level of secretory protein in the control group was 2.34+-0.41 μg/ml (mean+-S.E.M.). The level in cord blood was slightly lower (0.74+-0.26 μg/ml), while the level in patients with ovarian carcinoma was significantly increased (12.67+-1.43 μg/ml). Pregnant women have increasingly secretory protein levels with increasing length of gestation (5.86+-2.02, 11.55+-1.30 and 17.00+-1.16 μg/ml for the first, second and third trimesters, respectively. (Auth.)

  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. Protein-mediated surface structuring in biomembranes

    Directory of Open Access Journals (Sweden)

    Maggio B.

    2005-01-01

    Full Text Available The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein, integral (Folch-Lees proteolipid protein and amphitropic (c-Fos and c-Jun proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase, in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

  7. Biomimetic surface coatings from modular amphiphilic proteins

    Science.gov (United States)

    Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

    2010-03-01

    Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

  8. Surface Chloride Levels in Colorado Structural Concrete

    Science.gov (United States)

    2018-01-01

    This project focused on the chloride-induced corrosion of reinforcing steel in structural concrete. The primary goal of this project is to analyze the surface chloride concentration level of the concrete bridge decks throughout Colorado. The study in...

  9. Identification and characterization of the surface proteins of Clostridium difficile

    International Nuclear Information System (INIS)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated

  10. Level crossing analysis of growing surfaces

    International Nuclear Information System (INIS)

    Shahbazi, F; Sobhanian, S; Tabar, M Reza Rahimi; Khorram, S; Frootan, G R; Zahed, H

    2003-01-01

    We investigate the average frequency of positive slope ν + α , crossing the height α = h - h-bar in the surface growing processes. The exact level crossing analysis of the random deposition model and the Kardar-Parisi-Zhang equation in the strong coupling limit before creation of singularities is given

  11. The Electrophoretic Mobility of Proteins near Surfaces

    Science.gov (United States)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  12. Surface-to-surface registration using level sets

    DEFF Research Database (Denmark)

    Hansen, Mads Fogtmann; Erbou, Søren G.; Vester-Christensen, Martin

    2007-01-01

    This paper presents a general approach for surface-to-surface registration (S2SR) with the Euclidean metric using signed distance maps. In addition, the method is symmetric such that the registration of a shape A to a shape B is identical to the registration of the shape B to the shape A. The S2SR...... problem can be approximated by the image registration (IR) problem of the signed distance maps (SDMs) of the surfaces confined to some narrow band. By shrinking the narrow bands around the zero level sets the solution to the IR problem converges towards the S2SR problem. It is our hypothesis...... that this approach is more robust and less prone to fall into local minima than ordinary surface-to-surface registration. The IR problem is solved using the inverse compositional algorithm. In this paper, a set of 40 pelvic bones of Duroc pigs are registered to each other w.r.t. the Euclidean transformation...

  13. Decommissioning high-level waste surface facilities

    International Nuclear Information System (INIS)

    1978-04-01

    The protective storage, entombment and dismantlement options of decommissioning a High-Level Waste Surface Facility (HLWSF) was investigated. A reference conceptual design for the facility was developed based on the designs of similar facilities. State-of-the-art decommissioning technologies were identified. Program plans and cost estimates for decommissioning the reference conceptual designs were developed. Good engineering design concepts were on the basis of this work identified

  14. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  15. Surface modification of protein enhances encapsulation in chitosan nanoparticles

    Science.gov (United States)

    Koyani, Rina D.; Andrade, Mariana; Quester, Katrin; Gaytán, Paul; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2018-04-01

    Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

  16. Identification of surface proteins in Enterococcus faecalis V583

    Directory of Open Access Journals (Sweden)

    Eijsink Vincent GH

    2011-03-01

    Full Text Available Abstract Background Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31 or to be secreted (5. Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5 and surface (31 proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

  17. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    Science.gov (United States)

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  18. Characterization of the Eimeria maxima sporozoite surface protein IMP1.

    Science.gov (United States)

    Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

    2015-07-30

    The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. Published by Elsevier B.V.

  19. Competitive Protein Adsorption on Polysaccharide and Hyaluronate Modified Surfaces

    Science.gov (United States)

    Ombelli, Michela; Costello, Lauren; Postle, Corinne; Anantharaman, Vinod; Meng, Qing Cheng; Composto, Russell J.; Eckmann, David M.

    2011-01-01

    We measured adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) onto six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and streptococcus zooepidemicus. Film thickness and surface morphology depended on HA molecular weight and concentration. BSA coverage was enhanced on surfaces upon competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of hyaluronic acid utilized. With changing bulk protein concentration from 20 to 40 µg ml−1 for each species, Fg coverage on silicon increased by 4×, whereas both BSA and Fg adsorption on dextran and HA were far less dependent of protein bulk concentration. PMID:21623481

  20. SURF'S UP! – Protein classification by surface comparisons

    Indian Academy of Sciences (India)

    Prakash

    encounter large protein families with only a few members of ... server for analysis of functional relationships in protein families, as inferred from protein surface maps comparison ... features, SURF'S UP! can work with models obtained from comparative modelling. ... 1997) or, if the user is confident in the quality of automated.

  1. Directed supramolecular surface assembly of SNAP-tag fusion proteins

    NARCIS (Netherlands)

    Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, H.; Schenkel, J.H.; Huskens, J.; Ravoo, B.J.; Jonkheijm, P.; Brunsveld, L.

    2012-01-01

    Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

  2. Directed Supramolecular Surface Assembly of SNAP-tag Fusion Proteins

    NARCIS (Netherlands)

    Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, Hoang D.; Schenkel, J.H.; Huskens, Jurriaan; Ravoo, B.J.; Jonkheijm, Pascal; Brunsveld, Luc

    2012-01-01

    Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

  3. VASCo: computation and visualization of annotated protein surface contacts

    Directory of Open Access Journals (Sweden)

    Thallinger Gerhard G

    2009-01-01

    Full Text Available Abstract Background Structural data from crystallographic analyses contain a vast amount of information on protein-protein contacts. Knowledge on protein-protein interactions is essential for understanding many processes in living cells. The methods to investigate these interactions range from genetics to biophysics, crystallography, bioinformatics and computer modeling. Also crystal contact information can be useful to understand biologically relevant protein oligomerisation as they rely in principle on the same physico-chemical interaction forces. Visualization of crystal and biological contact data including different surface properties can help to analyse protein-protein interactions. Results VASCo is a program package for the calculation of protein surface properties and the visualization of annotated surfaces. Special emphasis is laid on protein-protein interactions, which are calculated based on surface point distances. The same approach is used to compare surfaces of two aligned molecules. Molecular properties such as electrostatic potential or hydrophobicity are mapped onto these surface points. Molecular surfaces and the corresponding properties are calculated using well established programs integrated into the package, as well as using custom developed programs. The modular package can easily be extended to include new properties for annotation. The output of the program is most conveniently displayed in PyMOL using a custom-made plug-in. Conclusion VASCo supplements other available protein contact visualisation tools and provides additional information on biological interactions as well as on crystal contacts. The tool provides a unique feature to compare surfaces of two aligned molecules based on point distances and thereby facilitates the visualization and analysis of surface differences.

  4. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

  5. Protein C activity and antigen levels in childhood

    NARCIS (Netherlands)

    van Teunenbroek, A.; Peters, M.; Sturk, A.; Borm, J. J.; Breederveld, C.

    1990-01-01

    Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined

  6. Trichomonas vaginalis surface proteins: a view from the genome

    DEFF Research Database (Denmark)

    Hirt, R. P.; Noel, C. J.; Sicheritz-Pontén, Thomas

    2007-01-01

    Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa....... However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T...

  7. Hydration dynamics near a model protein surface

    International Nuclear Information System (INIS)

    Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

    2003-01-01

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces

  8. Enhanced microcontact printing of proteins on nanoporous silica surface

    Energy Technology Data Exchange (ETDEWEB)

    Blinka, Ellen; Hu Ye; Gopal, Ashwini; Hoshino, Kazunori; Lin, Kevin; Zhang, John X J [Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78758 (United States); Loeffler, Kathryn; Liu Xuewu; Ferrari, Mauro, E-mail: John.Zhang@engr.utexas.edu [Department of Nanomedicine and Biomedical Engineering, University of Texas Health Science Service, Houston, TX 77031 (United States)

    2010-10-15

    We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

  9. Cleaning of biomaterial surfaces: protein removal by different solvents.

    Science.gov (United States)

    Kratz, Fabian; Grass, Simone; Umanskaya, Natalia; Scheibe, Christian; Müller-Renno, Christine; Davoudi, Neda; Hannig, Matthias; Ziegler, Christiane

    2015-04-01

    The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Radio-iodinated surface proteins of electrophoretically separated rat lymphocytes

    International Nuclear Information System (INIS)

    Jilg, W.; Hannig, K.; Zeiller, K.

    1980-01-01

    Rat thymocytes and lymph node cells were separated into three T and one B subpopulation by means of free flow electrophoresis. The surface proteins of the separated cells were labelled by lactoperoxidase catalysed radioiodination. Most of the label was demonstrated to be at the cell surface. Although the surface protein patterns of the four lamphocyte subpopulations were rather similar, distinctive differences could be found. B cells had six labelled proteins which seemed to be absent in the other cells. In the T cell group three protein bands were identified, each with specificity for peripheral T cells, thymocytes and all T cells respectively. Four other proteins were found which showed quantitative differences between the four cell groups. (orig.) [de

  11. Modulating Protein Adsorption on Oxygen Plasma Modified Polysiloxane Surfaces

    International Nuclear Information System (INIS)

    Marletta, G.

    2006-01-01

    In the present paper we report the study on the adsorption behaviour of three model globular proteins, Human Serum Albumin, Lactoferrin and Egg Chicken Lysozyme onto both unmodified surfaces of a silicon-based polymer and the corresponding plasma treated surfaces. In particular, thin films of hydrophobic polysiloxane (about 90 degree of static water contact angle, WCA) were converted by oxygen plasma treatment at reduced pressure into very hydrophilic phases of SiOx (WCA less than 5 degree). The kinetics of protein adsorption processes were investigated by QCM-D technique, while the chemical structure and topography of the protein adlayer have been studied by Angular resolved-XPS and AFM respectively. It turned out that Albumin and Lysozyme exhibited the opposite preferential adsorption respectively onto the hydrophobic and hydrophilic surfaces, while Lactoferrin did not exhibit significant differences. The observed protein behaviour are discussed both in terms of surface-dependent parameters, including surface free energy and chemical structure, and in terms of protein-dependent parameters, including charge as well as the average molecular orientation in the adlayers. Finally, some examples of differential adsorption behaviour of the investigated proteins are reported onto nanopatterned polysiloxane surfaces consisting of hydrophobic nanopores surrounded by hydrophilic (plasma-treated) matrix and the reverse

  12. Role of sperm surface proteins in reproduction

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Maňásková, Pavla; Davidová, Nina; Tichá, M.; Pěknicová, Jana

    2009-01-01

    Roč. 30, Supplement (2009), s. 63-64 ISSN 0196-3635. [9th International Congress of Andrology. 07.03.2009-10.03.2009, Barcelona] R&D Projects: GA MŠk(CZ) 1M06011; GA ČR(CZ) GA523/08/H064; GA ČR(CZ) GA303/06/0895 Institutional research plan: CEZ:AV0Z50520701 Keywords : boar seminal plasma proteins * spermadhesins * proteinase inhibitor * DQH * boar spermatozoa Subject RIV: CE - Biochemistry

  13. Effects of increasing dietary protein levels on growth, feed utilization ...

    African Journals Online (AJOL)

    Yomi

    2012-01-05

    Jan 5, 2012 ... The effect of different dietary protein levels on growth performance and on feed utilization of catfish. (Heterobranchus ... (Legendre, 1991) because of its taste, fast growth rate ..... diet containing 40% protein had high growth with low food intake and feed ... protein rate (45%) combined with a bad utilization of.

  14. Fermi level on hydrogen terminated diamond surfaces

    Czech Academy of Sciences Publication Activity Database

    Rezek, Bohuslav; Saurer, C.; Nebel, C. E.; Stutzmann, M.; Ristein, J.; Ley, L.; Snidero, E.; Bergonzo, P.

    2003-01-01

    Roč. 82, č. 14 (2003), s. 2266-2268 ISSN 0003-6951 EU Projects: European Commission(XE) HPRN-CT-1999-00139 Grant - others:DFC(DE) NE524-2 Institutional research plan: CEZ:AV0Z1010914 Keywords : atomic force microscope (AFM) * Kelvin probe experiments * diamond surface Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 4.049, year: 2003

  15. Protein-protein interaction site predictions with three-dimensional probability distributions of interacting atoms on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Ching-Tai Chen

    Full Text Available Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins and were tested on an independent dataset (consisting of 142 proteins. The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted

  16. Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

    Science.gov (United States)

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

  17. Extractable Bacterial Surface Proteins in Probiotic–Host Interaction

    Directory of Open Access Journals (Sweden)

    Fillipe L. R. do Carmo

    2018-04-01

    Full Text Available Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp, this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs, and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

  18. Changes in Serum Proteins and Creatinine levels in HIV Infected ...

    African Journals Online (AJOL)

    This study examined the level of total serum proteins and globulins in HIV infected Nigerians. 64 patients with HIV infection and 10 apparently healthy subjects were recruited from 3 hospitals in Lagos Metropolis. They were examined for the presence of TB and malaria. Serum total protein, albumin and creatinine levels ...

  19. Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins

    International Nuclear Information System (INIS)

    Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

    1987-01-01

    Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by 125 I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml

  20. Competitive protein adsorption to polymer surface from human serum

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Larsen, Niels Bent

    2008-01-01

    Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using...... different radioisotopes, albumin and Immunoglobulin G (IgG) adsorption has been monitored simultaneously during competitive adsorption processes, which to our knowledge has not been reported in the literature before. Results show that albumin and IgG adsorption is dependent on adsorption time...... and on the presence and concentration of other proteins in bulk solutions during adsorption. Generally, lower albumin and IgG adsorption was observed on the modified and more hydrophilic polymer surfaces, but otherwise the modified and unmodified polymer surfaces showed the same adsorption characteristics....

  1. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-08

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  2. Surface charge effects in protein adsorption on nanodiamonds.

    Science.gov (United States)

    Aramesh, M; Shimoni, O; Ostrikov, K; Prawer, S; Cervenka, J

    2015-03-19

    Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.

  3. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  4. Self-assembling triblock proteins for biofunctional surface modification

    Science.gov (United States)

    Fischer, Stephen E.

    Despite the tremendous promise of cell/tissue engineering, significant challenges remain in engineering functional scaffolds to precisely regulate the complex processes of tissue growth and development. As the point of contact between the cells and the scaffold, the scaffold surface plays a major role in mediating cellular behaviors. In this dissertation, the development and utility of self-assembling, artificial protein hydrogels as biofunctional surface modifiers is described. The design of these recombinant proteins is based on a telechelic triblock motif, in which a disordered polyelectrolyte central domain containing embedded bioactive ligands is flanked by two leucine zipper domains. Under moderate conditions of temperature and pH, the leucine zipper end domains form amphiphilic alpha-helices that reversibly associate into homo-trimeric aggregates, driving hydrogel formation. Moreover, the amphiphilic nature of these helical domains enables surface adsorption to a variety of scaffold materials to form biofunctional protein coatings. The nature and stability of these coatings in various solution conditions, and their interaction with mammalian cells is the primary focus of this dissertation. In particular, triblock protein coatings functionalized with cell recognition sequences are shown to produce well-defined surfaces with precise control over ligand density. The impact of this is demonstrated in multiple cell types through ligand density-dependent cell-substrate interactions. To improve the stability of these physically self-assembled coatings, two covalent crosslinking strategies are described---one in which a zero-length chemical crosslinker (EDC) is utilized and a second in which disulfide bonds are engineered into the recombinant proteins. These targeted crosslinking approaches are shown to increase the stability of surface adsorbed protein layers with minimal effect on the presentation of many bioactive ligands. Finally, to demonstrate the versatility

  5. Protein sequences bound to mineral surfaces persist into deep time

    DEFF Research Database (Denmark)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa

    2016-01-01

    of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell......, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated...

  6. Protein signatures using electrostatic molecular surfaces in harmonic space

    Directory of Open Access Journals (Sweden)

    C. Sofia Carvalho

    2013-10-01

    Full Text Available We developed a novel method based on the Fourier analysis of protein molecular surfaces to speed up the analysis of the vast structural data generated in the post-genomic era. This method computes the power spectrum of surfaces of the molecular electrostatic potential, whose three-dimensional coordinates have been either experimentally or theoretically determined. Thus we achieve a reduction of the initial three-dimensional information on the molecular surface to the one-dimensional information on pairs of points at a fixed scale apart. Consequently, the similarity search in our method is computationally less demanding and significantly faster than shape comparison methods. As proof of principle, we applied our method to a training set of viral proteins that are involved in major diseases such as Hepatitis C, Dengue fever, Yellow fever, Bovine viral diarrhea and West Nile fever. The training set contains proteins of four different protein families, as well as a mammalian representative enzyme. We found that the power spectrum successfully assigns a unique signature to each protein included in our training set, thus providing a direct probe of functional similarity among proteins. The results agree with established biological data from conventional structural biochemistry analyses.

  7. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    Science.gov (United States)

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  8. Surface charge effects in protein adsorption on nanodiamonds

    Science.gov (United States)

    Aramesh, M.; Shimoni, O.; Ostrikov, K.; Prawer, S.; Cervenka, J.

    2015-03-01

    Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins

  9. Surface proteins and the formation of biofilms by Staphylococcus aureus.

    Science.gov (United States)

    Kim, Sung Joon; Chang, James; Rimal, Binayak; Yang, Hao; Schaefer, Jacob

    2018-03-01

    Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

    Science.gov (United States)

    Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

    2014-03-07

    The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

  11. Role of Streptococcus mutans surface proteins for biofilm formation

    Directory of Open Access Journals (Sweden)

    Michiyo Matsumoto-Nakano

    2018-02-01

    Full Text Available Summary: Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. An important virulence property of the bacterium is its ability to form biofilm known as dental plaque on tooth surfaces. In addition, this organism also produces glucosyltransferases, multiple glucan-binding proteins, protein antigen c, and collagen-binding protein, surface proteins that coordinate to produce dental plaque, thus inducing dental caries. Bacteria utilize quorum-sensing systems to modulate environmental stress responses. A major mechanism of response to signals is represented by the so called two-component signal transduction system, which enables bacteria to regulate their gene expression and coordinate activities in response to environmental stress. As for S. mutans, a signal peptide-mediated quorum-sensing system encoded by comCDE has been found to be a regulatory system that responds to cell density and certain environmental stresses by excreting a peptide signal molecule termed CSP (competence-stimulating peptide. One of its principal virulence factors is production of bacteriocins (peptide antibiotics referred to as mutacins. Two-component signal transduction systems are commonly utilized by bacteria to regulate bacteriocin gene expression and are also related to biofilm formation by S. mutans. Keywords: Streptococcus mutans, Surface proteins, Biofilm, Signal transduction

  12. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

    DEFF Research Database (Denmark)

    Boyd, A. R.; Burke, G. A.; Duffy, H.

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation...... to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role...

  13. Dietary protein level and performance of growing Baladi kids.

    Science.gov (United States)

    Abdelrahman, M M; Aljumaah, R S

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (Pkids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity.

  14. Deposition of heated whey proteins on a chromium oxide surface.

    NARCIS (Netherlands)

    Jeurnink, Th.; Verheul, M.; Cohen Stuart, M.A.; Kruif, de C.G.

    1996-01-01

    Whey protein solutions were given different heat treatments after which their deposition on a chromium oxide surface (the outer layer of stainless steel) was measured by reflectometry. The deposition was studied under controlled flow conditions by using a stagnation point flow configuration. The

  15. Effect Of Dietary Protein Levels On The Performance And Carcass ...

    African Journals Online (AJOL)

    Effect Of Dietary Protein Levels On The Performance And Carcass ... Nigerian Journal of Animal Production ... Response criteria such as weight gain and feed conversion ratio, among others, and carcass characteristics were measured.

  16. In vitro study of proteins surface activity by tritium probe

    International Nuclear Information System (INIS)

    Chernysheva, M.G.; Badun, G.A.

    2010-01-01

    A new technique for in vitro studies of biomacromolecules interactions, their adsorption at aqueous/organic liquid interfaces and distribution in the bulk of liquid/liquid systems was developed. The method includes (1) tritium labeling of biomolecules by tritium thermal activation method and (2) scintillation phase step with organic phase, which can be concerned as a model of cellular membrane. Two globular proteins lysozyme and human serum albumin tested. We have determined the conditions of tritium labeling when labeled by-products can be easy separated by means of dialysis and size-exclusion chromatography. Scintillation phase experiments were conducted for three types of organic liquids. Thus, the influences of the nature of organic phase on proteins adsorption and its distribution in the bulk of aqueous/organic liquid system were determined. It was found that proteins possess high surface activity at aqueous/organic liquid interface. Furthermore, values of hydrophobicity of globular proteins were found by the experiment. (author)

  17. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  18. Evaluation of protein adsorption onto a polyurethane nanofiber surface having different segment distributions

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Yuko; Koizumi, Gaku [Frontier Fiber Technology and Science, Graduate School of Engineering, University of Fukui (Japan); Sakamoto, Hiroaki, E-mail: hi-saka@u-fukui.ac.jp [Tenure-Track Program for Innovative Research, University of Fukui (Japan); Suye, Shin-ichiro [Frontier Fiber Technology and Science, Graduate School of Engineering, University of Fukui (Japan)

    2017-02-01

    Electrospinning is well known to be an effective method for fabricating polymeric nanofibers with a diameter of several hundred nanometers. Recently, the molecular-level orientation within nanofibers has attracted particular attention. Previously, we used atomic force microscopy to visualize the phase separation between soft and hard segments of a polyurethane (PU) nanofiber surface prepared by electrospinning. The unstretched PU nanofibers exhibited irregularly distributed hard segments, whereas hard segments of stretched nanofibers prepared with a high-speed collector exhibited periodic structures along the long-axis direction. PU was originally used to inhibit protein adsorption, but because the surface segment distribution was changed in the stretched nanofiber, here, we hypothesized that the protein adsorption property on the stretched nanofiber might be affected. We investigated protein adsorption onto PU nanofibers to elucidate the effects of segment distribution on the surface properties of PU nanofibers. The amount of adsorbed protein on stretched PU nanofibers was increased compared with that of unstretched nanofibers. These results indicate that the hard segment alignment on stretched PU nanofibers mediated protein adsorption. It is therefore expected that the amount of protein adsorption can be controlled by rotation of the collector. - Highlights: • The hard segments of stretched PU nanofibers exhibit periodic structures. • The adsorbed protein on stretched PU nanofibers was increased compared with PU film. • The hard segment alignment on stretched PU nanofibers mediated protein adsorption.

  19. Investigation of the molecular level interactions between mucins and food proteins: Spectroscopic, tribological and rheological studies

    OpenAIRE

    Celebioglu, Hilal Yilmaz; Chronakis, Ioannis S.; Lee, Seunghwan; Guðjónsdóttir, María

    2017-01-01

    The thesis investigated the structure and molecular-level interaction of β-lactoglobulin (BLG) and mucins, representing major components of the dairy products and saliva/digestion systems, respectively. Mucins are long glycoprotein molecules responsible for the gel nature of the mucous layer covers epithelial surfaces throughout the body. A literature review of the interactions of different mucin types and saliva mucins with several food proteins and food protein emulsions, as well as their f...

  20. Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

    Science.gov (United States)

    Coleman, David Andrew

    2009-01-01

    The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

  1. Heat shock proteins on the human sperm surface.

    Science.gov (United States)

    Naaby-Hansen, Soren; Herr, John C

    2010-01-01

    The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

  2. A Level Set Discontinuous Galerkin Method for Free Surface Flows

    DEFF Research Database (Denmark)

    Grooss, Jesper; Hesthaven, Jan

    2006-01-01

    We present a discontinuous Galerkin method on a fully unstructured grid for the modeling of unsteady incompressible fluid flows with free surfaces. The surface is modeled by embedding and represented by a levelset. We discuss the discretization of the flow equations and the level set equation...

  3. Evaluation of the levels of phthalate ester plasticizers in surface ...

    African Journals Online (AJOL)

    Evaluation of the levels of phthalate ester plasticizers in surface water of Ethiope ... Gas chromatography (GC) coupled with mass spectrometer (MS) was used to ... with their common use in plastic materials and other industrial chemicals.

  4. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

    Science.gov (United States)

    Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

    2014-01-01

    BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

  5. Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

    Science.gov (United States)

    MacLean, Lorna; Price, Helen; O'Toole, Peter

    2016-01-01

    Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

  6. Improved protein surface comparison and application to low-resolution protein structure data

    Directory of Open Access Journals (Sweden)

    Kihara Daisuke

    2010-12-01

    Full Text Available Abstract Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM, which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs. The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

  7. Improved protein surface comparison and application to low-resolution protein structure data.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2010-12-14

    Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

  8. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  9. Volumetric interpretation of protein adsorption: interfacial packing of protein adsorbed to hydrophobic surfaces from surface-saturating solution concentrations.

    Science.gov (United States)

    Kao, Ping; Parhi, Purnendu; Krishnan, Anandi; Noh, Hyeran; Haider, Waseem; Tadigadapa, Srinivas; Allara, David L; Vogler, Erwin A

    2011-02-01

    The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square or hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square or hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM. Copyright © 2010

  10. Investigation of the connection between the quality of protein, protein level and endogenous N excretion

    International Nuclear Information System (INIS)

    Koehler, R.; Gebhardt, G.

    1979-01-01

    The influence of various protein qualities as well as of different levels of protein on the amount of endogenous N excretion, metabolic fecal nitrogen (MFN) and endogenous urinary N (EUN) was determined in growing albino rats. The test rations were labelled with admixtures of 15 N-DL-methionine and 15 N-DL-lysine, respectively, or contained feed protein enriched with 15 N. EUN and MFN and their sum (the N maintenance requirement) showed the influence of the respective protein source and its dependence on the protein level. The endogenous N excretions showed an opposite tendency to the N balance; for high-quality protein feedstuffs with a high N balance (e.g. dried eggs) they are lower than for protein sources of inferior quality, with a low N-balance only (e.g. wheat gluten). Presumably this interaction of retention and maintenance is due to the complementary effect of exogenous and endogenous amino acids in the N and amino acid pool, respectively. Provided that the N dose and the live weight of the animals are comparable, the N balance appears to be more suitable as parameter for the description of the protein quality and the calculation of the protein utilisation than N retention, as the sum of N balance and the values of MFN and EUN (depending on the feedstuffs and the N level). (author)

  11. Human serum protein and C-reactive protein levels among HIV ...

    African Journals Online (AJOL)

    Human serum protein and C-reactive protein levels were determined among HIV patients visiting St Camillus Hospital, Uromi, Edo State, Nigeria, between January to March, 2013. Fifty (50) HIV patients (20 males; 30 females) and 50 control subjects (24 males; 26 females) were enrolled for this study. The clinical status of ...

  12. Dynamic, electronically switchable surfaces for membrane protein microarrays.

    Science.gov (United States)

    Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

    2006-02-01

    Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

  13. Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

    Science.gov (United States)

    Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

    2017-09-01

    Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

  14. Preventing protein adsorption from a range of surfaces using an aqueous fish protein extract

    DEFF Research Database (Denmark)

    Pillai, Saju; Arpanaei, Ayyoob; Meyer, Rikke L.

    2009-01-01

    We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed...

  15. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  16. Biological properties of Lactobacillus surface proteins 

    Directory of Open Access Journals (Sweden)

    Barbara Buda

    2013-04-01

    Full Text Available Lactobacillus, a genus of Gram-positive bacteria, includes many strains of probiotic microflora. Probiotics, by definition, are living microorganisms that exert beneficial effects on the host organism. The morphology and physiology of the Lactobacillus bacterial genus are described. The structure of the cell wall of Gram-positive bacteria is discussed. The surface S-layer of Lactobacillus composed of proteins (SLP with low molecular mass is presented. Cell surface proteins participating in the regulation of growth and survival of the intestinal epithelium cells are characterized. The influence of stress factors such as increased temperature, pH, and enzymes of gastric and pancreatic juice on SLP expression is described. The ability of binding of heavy metal ions by S-layer proteins is discussed. The characteristics of these structures, including the ability to adhere to epithelial cells, and the inhibition of invasion of pathogenic microflora of type Shigella, Salmonella, Escherichia coli and Clostridium and their toxins, are presented. 

  17. Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

    Science.gov (United States)

    Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

    2018-03-13

    Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

  18. Surface core-level shifts for simple metals

    DEFF Research Database (Denmark)

    Aldén, Magnus; Skriver, Hans Lomholt; Johansson, Börje

    1994-01-01

    screening, whereby a SCLS becomes equivalent to the surface segregation energy of a core-ionized atom, a quantity we obtain by separate bulk and surface impurity calculations. The results are in good agreement with experiment in most of those cases where the data originates from single-crystal measurements....... We discuss the surface shifts of the electrostatic potentials and the band centers in order to trace the microscopic origin of the SCLS in the simple metals and find that the anomalous subsurface core-level shifts in beryllium are caused by charge dipoles, which persist several layers into the bulk...

  19. Association of plasma protein C levels and coronary artery disease ...

    African Journals Online (AJOL)

    Several studies have shown the risk factor causes of coronary heart disease. In this study we tested the hypothesis that plasma protein C level might be used as a biomarker for coronary heart disease and myocardial infarction. The study included 60 men that were classified into 3 groups according to clinical examination; ...

  20. Serum protein and enzyme levels in rats following administration of ...

    African Journals Online (AJOL)

    The effects of caffeinated and non-caffeinated paracetamol administration, with or without vitamins A and E supplementation on the protein and enzyme levels in Wistar albino rats were investigated using cafeinated paracetamol and paracetamol as caffeinated and non-caffeinated paracetamol respectively, and water ...

  1. Effect Of Different Protein Levels on The Performance Of Growing ...

    African Journals Online (AJOL)

    T4 was superior to other treatments in terms of feed to gain ratio, efficiency of feed utilization and reproductive performance. Snails in T1 with 10% CP failed to lay eggs and had the least developed reproductive system. A diet of 15-20% C. P is therefore recommended for growing snails. Key words: Protein levels, snails, ...

  2. Interactive effect of dietary protein level and zilpaterol hydrochloride ...

    African Journals Online (AJOL)

    Bonsmara type steers were used to determine the effect of dietary zilpaterol hydrochloride (ZH) in combination with different dietary crude protein (CP) levels (100, 120 and 140 g CP/kg) on growth performance and meat quality. Treatment groups (T) consisted of 12 steers each. T1 – 100 g CP/kg + 0.15 mg ZH/kg live weight ...

  3. Effect Of Crude Protein Levels And Follicle Stimulation On Egg ...

    African Journals Online (AJOL)

    Two groups received 16% crude protein (CP) level diets and the other two groups, 32%. One each of the two groups received follicle stimulation, induced by administration of Clomifene citrate (1.5mg/kg) via cathetered 5ml syringe through the 10week experimental period, with feed and water offered ad libitum.

  4. Effect of dietary crude protein level on the performance and ...

    African Journals Online (AJOL)

    ویرایه

    2013-06-26

    Jun 26, 2013 ... The effects of increasing dietary levels of crude protein (CP) on growth, feed intake, feed efficiency and nutrient apparent ... matter intake (DMI) than the kids fed with 10.5, 12.8, .... Food and Agriculture Organization. Database ...

  5. Protein adsorption at nanopatterned surfaces studied by QCM-D and SPR

    DEFF Research Database (Denmark)

    Kristensen, Stine; Pedersen, Gitte Albinus; Nejsum, Lene Niemann

    2013-01-01

    This paper presents the use of the quartz microbalance with dissipation combined with surface plasmon resonance to probe protein adsorption at nanopatterned surfaces. Three different types of adsorbing materials, representing rigid discrete nanoparticles, dense protein films and soft low density ...

  6. Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

    Science.gov (United States)

    MacLaughlin, Christina M.

    Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

  7. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Sasnauskas Kęstutis

    2011-05-01

    Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

  8. The Cutoff Level for Urine Protein in Urine Immunofixation Electrophoresis.

    Science.gov (United States)

    Ellidag, Hamit Yasar; Curek, Gulten; Eren, Esin; Aydin, Ozgur; Yilmaz, Necat

    2015-01-01

    Immunofixation electrophoresis (IFE) maintains its importance in diagnosing monoclonal gammopathies. In particular, urine IFE detects free light chains (FLC) in urine samples even at low concentrations and offers higher sensitivity compared to serum electrophoresis and serum IFE. The aim of the present study was to determine the place and significance of quantitative urinary protein measurement before IFE in interpreting the results of subsequent IFE and to determine the most appropriate protein concentrations for the appearance of bands. The records of a total of 600 patients, who underwent screening for Bence Jones proteinuria using IFE on 24-hour urine, were retrospectively reviewed. Urine IFE was performed using Helena SAS-I and SAS-I devices. The total protein concentration in the urine was quantitatively determined by the Pyrogallol red method, and the urine albumin level was determined using the immunoturbidimetric method. These analyses were measured on an Olympus/Beckmann AU5800. The evaluation of IFE results revealed that 311 patients had normal results, 108 patients had monoclonal bands, five patients had biclonal bands, 28 had polyclonal bands, and 148 patients had various degrees of proteinuria. ROC curves were created in order to determine the most appropriate urinary protein and albumin levels to observe bands in IFE. Accordingly, urine baseline protein level (mg/dL) showed the highest AUC value (cutoff value: 19.4 mg/dL, sensitivity: 92%, specificity: 98.2%, AUC: 0.972). The present study showed that quantitative protein measurement before IFE eliminated the disadvantages associated with the IFE method and its interpretation.

  9. Proteins in Complementary Food: What Is the Healthiest Level?

    Directory of Open Access Journals (Sweden)

    О. K. Netrebenko

    2015-01-01

    Full Text Available Adequate protein consumption in infants is a heavily debated issue. First, it is related to the formation of a new scientific field — “Infant prerequisites of man’s wellness and illness,” which directly indicates that excessive intake of proteins during infancy has long-term consequences and greatly contributes to obesity and chronic infectious diseases in adults; second, it is related to new technologies, which improve the protein component of infant formulas and bring them at par with breast milk in terms of quality and quantity. High protein consumption is related to bottle feeding, because starter and further infant formulas are richer in protein than breast milk. Protein-rich menus trigger production of insulinogenic amino acids, insulin and the insulin-like growth factor (IGF-1. High IFTcombined with branched-chain amino acids (leucine, valine, isoleucine, threonine activates a set of signalling molecules (mTOR, which are responsible for integrating metabolic and immune response. Repeated activation of mTOR coupled with regular intake of high-protein infant formulas causes health issues in adulthood. Diseases like diabetes type 2, obesity, arterial hypertension, cancer (particularly prostatic cancer, are related to overactivation of the mTOR signalling molecule complex. Intensive consumption of milk in today’s world is the key mTOR activator contributing to an increased risk of lifestyle diseases and triggering the mechanism of their development. The progressing infant formula industry allows to cut protein levels in starter and further infant formulas down to 12 g/l and, respectively, lower the risk of non-infectious diseases in adulthood. 

  10. Surface peptide mapping of protein I and protein III of four strains of Neisseria gonorrhoeae

    International Nuclear Information System (INIS)

    Judd, R.C.

    1982-01-01

    Whole cells and isolated outer membranes (OMs) of four strains of gonococci were surface radioiodinated with either lactoperoxidase or Iodogen (Pierce Chemical Co., Rockford, Ill.). These preparations were solubilized in sodium dodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and digested with alpha-chymotrypsin, and the resultant 125 I-peptide fragments were resolved by high-voltage electrophoresis and thin-layer chromatography (i.e., surface peptide mapping). Radioemitting peptidic fragments were visualized by autoradiography. Results demonstrated that the PI molecule of each gonococcal strain studied had unique iodinatable peptides exposed on the surface of whole cells and OMs, whereas PIIIs appeared to have the same portion of the molecule exposed on the surface of bacteria or OMs, regardless of the gonococcal strain from which they were isolated. Many more radiolabeled peptides were seen in surface peptide maps of PIs from radiolabeled OMs than in those from radioiodinated whole cells, whereas different peptidic fragments were seen in the surface peptide maps of PIIIs from radiolabeled OMs than were seen in those from radiolabeled whole cells. These data suggest that PI may contribute strain-specific antigenic determinants and PIII may contribute cross-reactive determinants and that the surface exposure of PI and PIII is different in isolated OMs than in the OM of intact gonococci

  11. Genetic and environmental influences of surfactant protein D serum levels

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Hjelmborg, Jacob v. B.; Kyvik, Kirsten Ohm

    2006-01-01

    in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass...... defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located...

  12. Genetic and environmental influences of surfactant protein D serum levels

    DEFF Research Database (Denmark)

    Sorensen, G.L.; Hjelmborg, J.V.; Kyvik, K.O.

    2006-01-01

    defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located...... in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass...

  13. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface

    KAUST Repository

    Capriotti, Anna Laura; Caracciolo, Giulio; Cavaliere, Chiara; Crescenzi, Carlo; Pozzi, Daniela; Laganà , Aldo

    2011-01-01

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected

  14. Total proteins and protein fractions levels in pregnant rats subjected to whole-body gamma irradiation

    International Nuclear Information System (INIS)

    Mansour, M.A.; Roushdy, H.M.; Mazhar, F.M.; Abu-Gabal, H.A.

    1986-01-01

    A total number of 180 mature rats (120 females and 60 males) weighing from 120-140 g were used to study the effect of two doses (2 and 4 Gy) whole-body gamma irradiation on the level of total protein and protein fractions in serum of pregnant rats during the period of organogenesis. It was found that the levels of total protein, albumin and gamma globulins significantly decreased according to the doses of exposure. The levels of alpha and beta globulins significantly increased more in the serum of rats exposed to 2 Gy than in rats exposed to 4 Gy. The level of A/G ratio significantly decreased more in the serum of rats exposed to 2Gy than in those exposed to 4 Gy

  15. Automatic Measurement of Low Level Contamination on Concrete Surfaces

    International Nuclear Information System (INIS)

    Tachibana, M.; Itoh, H.; Shimada, T.; Yanagihara, S.

    2002-01-01

    Automatic measurement of radioactivity is necessary for considering cost effectiveness in final radiological survey of building structures in decommissioning nuclear facilities. The RAPID (radiation measuring pilot device for surface contamination) was developed to be applied to automatic measurement of low level contamination on concrete surfaces. The RAPID has a capability to measure contamination with detection limit of 0.14 Bq/cm2 for 60Co in 30 seconds of measurement time and its efficiency is evaluated to be 5 m2/h in a normal measurement option. It was confirmed that low level contamination on concrete surfaces could be surveyed by the RAPID efficiently compared with direct measurement by workers through its actual application

  16. Origin of metallic surface core-level shifts

    DEFF Research Database (Denmark)

    Aldén, Magnus; Skriver, Hans Lomholt; Abrikosov, I. A.

    1995-01-01

    The unique property of the open 4f energy shell in the lanthanide metals is used to show that the initial-state energy shift gives an insufficient description of surface core-level shifts. Instead a treatment, which fully includes the final-state screening, account for the experimentally observed...

  17. Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Boesen, Thomas; Hjernø, Karin

    1999-01-01

    present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used...

  18. Prediction of antigenic epitopes on protein surfaces by consensus scoring

    Directory of Open Access Journals (Sweden)

    Zhang Chi

    2009-09-01

    Full Text Available Abstract Background Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance. Results We present a new antigen Epitope Prediction method, which uses ConsEnsus Scoring (EPCES from six different scoring functions - residue epitope propensity, conservation score, side-chain energy score, contact number, surface planarity score, and secondary structure composition. Applied to unbounded antigen structures from an independent test set, EPCES was able to predict antigenic eptitopes with 47.8% sensitivity, 69.5% specificity and an AUC value of 0.632. The performance of the method is statistically similar to other published methods. The AUC value of EPCES is slightly higher compared to the best results of existing algorithms by about 0.034. Conclusion Our work shows consensus scoring of multiple features has a better performance than any single term. The successful prediction is also due to the new score of residue epitope propensity based on atomic solvent accessibility.

  19. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    Science.gov (United States)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  20. Surface proteins of bacteria of the genus Bifidobacterium 

    Directory of Open Access Journals (Sweden)

    Ewa Dylus

    2013-05-01

    Full Text Available Beneficial effects due to the presence of probiotic bacteria of the genus Bifidobacterium in the human intestinal tract are still an interesting object of study. So far activities have been confirmed of bifidobacteria in stimulation of the host immune system, stimulation of tumor cell apoptosis, improvement of bowel motility, alleviation of symptoms of lactose intolerance, cholesterol lowering capacity, prevention and treatment of diarrhea and irritable bowel syndrome, alleviation of allergy or atopic dermatitis, maintenance of homeostasis of the intestine, and stimulation of the development of normal intestinal microflora in infants. A multitude of therapeutic properties encourages researchers to investigate the possibility of using the potential of Bifidobacterium in the prevention and treatment of other conditions such as rheumatoid arthritis and depression. Although it is known that the beneficial effects are due to intestinal mucosal colonization by these bacteria, the cell components responsible for the colonization are still not determined. In addition to the beneficial effects of probiotic administration, there were also negative effects including sepsis. Therefore research has been directed to identify specific components of Bifidobacterium responsible for probiotic effects. Currently researchers are focused on identifying, isolating and evaluating the properties of surface proteins that are probably involved in the adhesion of bacterial cells to the intestinal epithelium, improving colonization. This paper is an overview of current knowledge on Bifidobacterium surface proteins. The ways of transport and anchoring proteins in Gram-positive bacterial cells, the assembly of cell wall, and a description of the genus Bifidobacterium are presented.

  1. Low copper and high manganese levels in prion protein plaques

    Science.gov (United States)

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  2. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

    Science.gov (United States)

    Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

    2012-12-14

    C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

  3. Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein*

    Science.gov (United States)

    Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M.; Di Guilmi, Anne Marie; Frachet, Philippe

    2012-01-01

    C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (KD = 0.34–2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

  4. Effect of feeding different dietary protein levels on reproductive ...

    African Journals Online (AJOL)

    A feeding trial was conducted to evaluate effects of feeding different dietary protein levels on reproductive biology of African mud catfish under hapa system. Catfish fingerlings (mean body weight (4.50± 0.01g) and total length (8.0±0.2cm) were randomly stocked at 20 fish per hapa (1m3). Five experimental diets with crude ...

  5. Investigation of the molecular level interactions between mucins and food proteins: Spectroscopic, tribological and rheological studies

    DEFF Research Database (Denmark)

    Celebioglu, Hilal Yilmaz

    The thesis investigated the structure and molecular-level interaction of β-lactoglobulin (BLG) and mucins, representing major components of the dairy products and saliva/digestion systems, respectively. Mucins are long glycoprotein molecules responsible for the gel nature of the mucous layer covers...... epithelial surfaces throughout the body. A literature review of the interactions of different mucin types and saliva mucins with several food proteins and food protein emulsions, as well as their functional properties related to the food oral processing is presented at the first chapter of the thesis (Paper...... V). Most of the studies suggest an electrostatic attraction between positively charged food proteins with negatively charged moieties of mucins (mainly on glycosylated region of mucins). The structural changes occurring during the interaction between BLG, the major whey protein, and bovine...

  6. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © 2014 Wiley Periodicals, Inc.

  7. Evaluation of hydrogen and oxygen impurity levels on silicon surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Kenny, M J; Wielunski, L S; Netterfield, R P; Martin, P J; Leistner, A [Commonwealth Scientific and Industrial Research Organisation (CSIRO), Lindfield, NSW (Australia). Div. of Applied Physics

    1997-12-31

    This paper reports on surface analytical techniques used to quantify surface concentrations of impurities such as oxygen and hydrogen. The following analytical techniques were used: Rutherford and Backscattering, elastic recoil detection, time-of-flight SIMS, spectroscopic ellipsometry, x-ray photoelectron spectroscopy. The results have shown a spread in thickness of oxide layer, ranging from unmeasurable to 1.6 nm. The data must be considered as preliminary at this stage, but give some insight into the suitability of the techniques and a general idea of the significance of impurities at the monolayer level. These measurements have been carried out on a small number of silicon surfaces both semiconductor grade <111> crystalline material and silicon which has been used in sphere fabrication. 5 refs., 1 fig.

  8. Evaluation of hydrogen and oxygen impurity levels on silicon surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Kenny, M.J.; Wielunski, L.S.; Netterfield, R.P.; Martin, P.J.; Leistner, A. [Commonwealth Scientific and Industrial Research Organisation (CSIRO), Lindfield, NSW (Australia). Div. of Applied Physics

    1996-12-31

    This paper reports on surface analytical techniques used to quantify surface concentrations of impurities such as oxygen and hydrogen. The following analytical techniques were used: Rutherford and Backscattering, elastic recoil detection, time-of-flight SIMS, spectroscopic ellipsometry, x-ray photoelectron spectroscopy. The results have shown a spread in thickness of oxide layer, ranging from unmeasurable to 1.6 nm. The data must be considered as preliminary at this stage, but give some insight into the suitability of the techniques and a general idea of the significance of impurities at the monolayer level. These measurements have been carried out on a small number of silicon surfaces both semiconductor grade <111> crystalline material and silicon which has been used in sphere fabrication. 5 refs., 1 fig.

  9. Trace-level mercury removal from surface water

    International Nuclear Information System (INIS)

    Klasson, K.T.; Bostick, D.T.

    1998-01-01

    Many sorbents have been developed for the removal of mercury and heavy metals from waters; however, most of the data published thus far do not address the removal of mercury to the target levels represented in this project. The application to which these sorbents are targeted for use is the removal of mercury from microgram-per-liter levels to low nanogram-per-liter levels. Sorbents with thiouronium, thiol, amine, sulfur, and proprietary functional groups were selected for these studies. Mercury was successfully removed from surface water via adsorption onto Ionac SR-4 and Mersorb resins to levels below the target goal of 12 ng/L in batch studies. A thiol-based resin performed the best, indicating that over 200,000 volumes of water could be treated with one volume of resin. The cost of the resin is approximately $0.24 per 1,000 gal of water

  10. Sea level: measuring the bounding surfaces of the ocean

    Science.gov (United States)

    Tamisiea, Mark E.; Hughes, Chris W.; Williams, Simon D. P.; Bingley, Richard M.

    2014-01-01

    The practical need to understand sea level along the coasts, such as for safe navigation given the spatially variable tides, has resulted in tide gauge observations having the distinction of being some of the longest instrumental ocean records. Archives of these records, along with geological constraints, have allowed us to identify the century-scale rise in global sea level. Additional data sources, particularly satellite altimetry missions, have helped us to better identify the rates and causes of sea-level rise and the mechanisms leading to spatial variability in the observed rates. Analysis of all of the data reveals the need for long-term and stable observation systems to assess accurately the regional changes as well as to improve our ability to estimate future changes in sea level. While information from many scientific disciplines is needed to understand sea-level change, this review focuses on contributions from geodesy and the role of the ocean's bounding surfaces: the sea surface and the Earth's crust. PMID:25157196

  11. Sea level: measuring the bounding surfaces of the ocean.

    Science.gov (United States)

    Tamisiea, Mark E; Hughes, Chris W; Williams, Simon D P; Bingley, Richard M

    2014-09-28

    The practical need to understand sea level along the coasts, such as for safe navigation given the spatially variable tides, has resulted in tide gauge observations having the distinction of being some of the longest instrumental ocean records. Archives of these records, along with geological constraints, have allowed us to identify the century-scale rise in global sea level. Additional data sources, particularly satellite altimetry missions, have helped us to better identify the rates and causes of sea-level rise and the mechanisms leading to spatial variability in the observed rates. Analysis of all of the data reveals the need for long-term and stable observation systems to assess accurately the regional changes as well as to improve our ability to estimate future changes in sea level. While information from many scientific disciplines is needed to understand sea-level change, this review focuses on contributions from geodesy and the role of the ocean's bounding surfaces: the sea surface and the Earth's crust. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  12. Surface drainage in leveled land: Implication of slope

    Directory of Open Access Journals (Sweden)

    Antoniony S. Winkler

    Full Text Available ABSTRACT In the lowlands of Rio Grande do Sul, land leveling is mostly carried out with no slope for the purpose of rice production. In this environment, soils with a low hydraulic conductivity are predominant owing to the presence of a practically impermeable B-horizon near the surface. Land leveling leads to soil accommodation resulting in the formation of depressions where water accumulates after heavy rainfalls, subsequently leading to problems with crops implanted in succession to rice, such as soybeans. The objective of this research was to quantify the areas and volumes of water accumulation in soil as a function of the slope of land leveling. Five typical leveled lowland areas were studied as a part of this research. The original areas presented slopes of 0, 0.20, 0.25, 0.28 and 0.40%, which were used to generate new digital elevation models with slopes between 0 and 0.5%. These newly generated digital models were used to map the depressions with surface water storage. In conclusion, land leveling with slopes higher than 0.1% is recommended to minimize problems with superficial water storage in rice fields.

  13. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  14. New reactive polymer for protein immobilisation on sensor surfaces.

    Science.gov (United States)

    Kyprianou, Dimitris; Guerreiro, Antonio R; Chianella, Iva; Piletska, Elena V; Fowler, Steven A; Karim, Kal; Whitcombe, Michael J; Turner, Anthony P F; Piletsky, Sergey A

    2009-01-01

    Immobilisation of biorecognition elements 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. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the

  15. Experimental approach to controllably vary protein oxidation while minimizing electrode adsorption for boron-doped diamond electrochemical surface mapping applications.

    Science.gov (United States)

    McClintock, Carlee S; Hettich, Robert L

    2013-01-02

    Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent (i.e., hydroxyl radicals) for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate the oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins.

  16. C-reactive protein levels in patients with aggressive periodontitis.

    Science.gov (United States)

    Salzberg, Trang N; Overstreet, Benjamin T; Rogers, Jeffrey D; Califano, Joseph V; Best, Al M; Schenkein, Harvey A

    2006-06-01

    Sera from patients with periodontal infections contain elevated levels of C-reactive protein (CRP) compared to periodontally healthy individuals. Most studies to date have included patients with chronic periodontitis, and few investigators have studied CRP levels in subjects with aggressive periodontitis (AgP). The purpose of this study was to determine the relative levels of serum CRP in AgP patients and periodontally healthy subjects and to examine patients' characteristics that might account for intergroup differences. Serum samples were collected from 93 patients with generalized AgP (GAgP), from 97 patients with localized AgP (LAgP), and from 91 healthy controls (non-periodontitis [NP]). Periodontal examination consisted of plaque index, gingival index, probing depth, bleeding index, and attachment loss measurements. Current smoking was assessed by determination of serum cotinine levels by enzyme-linked immunosorbent assay (ELISA), and serum CRP levels were determined using a high-sensitivity ELISA assay. The three groups were significantly different from one another (P periodontal and demographic variables and current smoking, both mean probing depth and periodontal diagnosis remained correlated with CRP levels. Patients with AgP have statistically significant elevations in serum CRP levels compared to subjects without periodontitis. Elevated CRP in these subjects might represent a contribution of periodontal infections to systemic inflammation in relatively young individuals.

  17. C-Reactive Protein Levels in the Brugada Syndrome

    Directory of Open Access Journals (Sweden)

    Aimé Bonny

    2011-01-01

    Full Text Available Background. Inflammation in the Brugada syndrome (BrS and its clinical implication have been little studied. Aims. To assess the level of inflammation in BrS patients. Methods. All studied BrS patients underwent blood samples drawn for C-reactive protein (CRP levels at admission, prior to any invasive intervention. Patients with a previous ICD placement were controlled to exclude those with a recent (<14 days shock. We divided subjects into symptomatic (syncope or aborted sudden death and asymptomatic groups. In a multivariable analysis, we adjusted for significant variables (age, CRP ≥ 2 mg/L. Results. Fifty-four subjects were studied (mean age 45 ± 13 years, 49 (91% male. Twenty (37% were symptomatic. Baseline characteristics were similar in both groups. Mean CRP level was 1,4 ± 0,9 mg/L in asymptomatic and 2,4 ± 1,4 mg/L in symptomatic groups (P = .003. In the multivariate model, CRP concentrations ≥ 2 mg/L remained an independent marker for being symptomatic (P = .018; 95% CI: 1.3 to 19.3. Conclusion. Inflammation seems to be more active in symptomatic BrS. C-reactive protein concentrations ≥ 2 mg/L might be associated with the previous symptoms in BrS. The value of inflammation as a risk factor of arrhythmic events in BrS needs to be studied.

  18. Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach.

    Science.gov (United States)

    Reolon, Luciano Antonio; Martello, Carolina Lumertz; Schrank, Irene Silveira; Ferreira, Henrique Bunselmeyer

    2014-01-01

    The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae), the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.

  19. Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach.

    Directory of Open Access Journals (Sweden)

    Luciano Antonio Reolon

    Full Text Available The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae, the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.

  20. Optimized surface-slab excited-state muffin-tin potential and surface core level shifts

    International Nuclear Information System (INIS)

    Rundgren, J.

    2003-01-01

    An optimized muffin-tin (MT) potential for surface slabs with preassigned surface core-level shifts (SCLS's) is presented. By using the MT radii as adjustable parameters the model is able to conserve the definition of the SCLS with respect to the bulk and concurrently to generate a potential that is continuous at the MT radii. The model is conceived for elastic electron scattering in a surface slab with exchange-correlation interaction described by the local density approximation. The model employs two data bases for the self-energy of the signal electron (after Hedin and Lundqvist or Sernelius). The potential model is discussed in detail with two surface structures Be(101-bar0), for which SCLS's are available, and Cu(111)p(2x2)Cs, in which the close-packed radii of the atoms are extremely different. It is considered plausible that tensor LEED based on an optimized MT potential can be used for determining SCLS's

  1. Ab initio surface core-level shifts and surface segregation energies

    DEFF Research Database (Denmark)

    Aldén, Magnus; Skriver, Hans Lomholt; Johansson, Börje

    1993-01-01

    We have calculated the surface core-level energy shifts of the 4d and 5d transition metals by means of local-density theory and a Green’s-function technique based on the linear muffin-tin orbitals method. Final-state effects are included by treating the core-ionized atom as an impurity located in...

  2. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    Science.gov (United States)

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  3. Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

    Science.gov (United States)

    Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

    2006-07-01

    The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

  4. Levels of acute phase proteins remain stable after ischemic stroke

    Directory of Open Access Journals (Sweden)

    Paik Myunghee C

    2006-10-01

    Full Text Available Abstract Background Inflammation and inflammatory biomarkers play an important role in atherosclerosis and cardiovascular disease. Little information is available, however, on time course of serum markers of inflammation after stroke. Methods First ischemic stroke patients ≥40 years old had levels of high-sensitivity C-reactive protein (hsCRP, serum amyloid A (SAA, and fibrinogen measured in plasma samples drawn at 1, 2, 3, 7, 14, 21 and 28 days after stroke. Levels were log-transformed as needed, and parametric and non-parametric statistical tests were used to test for evidence of a trend in levels over time. Levels of hsCRP and SAA were also compared with levels in a comparable population of stroke-free participants. Results Mean age of participants with repeated measures (n = 21 was 65.6 ± 11.6 years, and 13 (61.9% were men, and 15 (71.4% were Hispanic. Approximately 75% of patients (n = 15 had mild strokes (NIH Stroke Scale score 0–5. There was no evidence of a time trend in levels of hsCRP, SAA, or fibrinogen for any of the markers during the 28 days of follow-up. Mean log(hsCRP was 1.67 ± 1.07 mg/L (median hsCRP 6.48 mg/L among stroke participants and 1.00 ± 1.18 mg/L (median 2.82 mg/L in a group of 1176 randomly selected stroke-free participants from the same community (p = 0.0252. Conclusion Levels of hsCRP are higher in stroke patients than in stroke-free subjects. Levels of inflammatory biomarkers associated with atherosclerosis, including hsCRP, appear to be stable for at least 28 days after first ischemic stroke.

  5. Effect of Crude Protein Levels in Concentrate and Concentrate Levels in Diet on Fermentation

    Directory of Open Access Journals (Sweden)

    Dinh Van Dung

    2014-06-01

    Full Text Available The effect of concentrate mixtures with crude protein (CP levels 10%, 13%, 16%, and 19% and diets with roughage to concentrate ratios 80:20, 60:40, 40:60, and 20:80 (w/w were determined on dry matter (DM and organic matter (OM digestibility, and fermentation metabolites using an in vitro fermentation technique. In vitro fermented attributes were measured after 4, 24, and 48 h of incubation respectively. The digestibility of DM and OM, and total volatile fatty acid (VFA increased whereas pH decreased with the increased amount of concentrate in the diet (p<0.001, however CP levels of concentrate did not have any influence on these attributes. Gas production reduced with increased CP levels, while it increased with increasing concentrate levels. Ammonia nitrogen (NH3-N concentration and microbial CP production increased significantly (p<0.05 by increasing CP levels and with increasing concentrate levels in diet as well, however, no significant difference was found between 16% and 19% CP levels. Therefore, 16% CP in concentrate and increasing proportion of concentrate up to 80% in diet all had improved digestibility of DM and organic matter, and higher microbial protein production, with improved fermentation characteristics.

  6. Hydrophobicity-driven self-assembly of protein and silver nanoparticles for protein detection using surface-enhanced Raman scattering.

    Science.gov (United States)

    Kahraman, Mehmet; Balz, Ben N; Wachsmann-Hogiu, Sebastian

    2013-05-21

    Surface-enhanced Raman scattering (SERS) is a promising analytical technique for the detection and characterization of biological molecules and structures. The role of hydrophobic and hydrophilic surfaces in the self-assembly of protein-metallic nanoparticle structures for label-free protein detection is demonstrated. Aggregation is driven by both the hydrophobicity of the surface as well as the charge of the proteins. The best conditions for obtaining a reproducible SERS signal that allows for sensitive, label-free protein detection are provided by the use of hydrophobic surfaces and 16 × 10(11) NPs per mL. A detection limit of approximately 0.5 μg mL(-1) is achieved regardless of the proteins' charge properties and size. The developed method is simple and can be used for reproducible and sensitive detection and characterization of a wide variety of biological molecules and various structures with different sizes and charge status.

  7. Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

    Directory of Open Access Journals (Sweden)

    Lee Sael

    2010-12-01

    Full Text Available Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

  8. Binding ligand prediction for proteins using partial matching of local surface patches.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2010-01-01

    Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

  9. Dynamics of gas-surface interactions atomic-level understanding of scattering processes at surfaces

    CERN Document Server

    Díez Muniño, Ricardo

    2013-01-01

    This book gives a representative survey of the state of the art of research on gas-surface interactions. It provides an overview of the current understanding of gas surface dynamics and, in particular, of the reactive and non-reactive processes of atoms and small molecules at surfaces. Leading scientists in the field, both from the theoretical and the experimental sides, write in this book about their most recent advances. Surface science grew as an interdisciplinary research area over the last decades, mostly because of new experimental technologies (ultra-high vacuum, for instance), as well as because of a novel paradigm, the ‘surface science’ approach. The book describes the second transformation which is now taking place pushed by the availability of powerful quantum-mechanical theoretical methods implemented numerically. In the book, experiment and theory progress hand in hand with an unprecedented degree of accuracy and control. The book presents how modern surface science targets the atomic-level u...

  10. AFM study of adsorption of protein A on a poly(dimethylsiloxane) surface

    International Nuclear Information System (INIS)

    Yu Ling; Lu Zhisong; Gan Ye; Liu Yingshuai; Li, C M

    2009-01-01

    In this paper, the morphology and kinetics of adsorption of protein A on a PDMS surface is studied by AFM. The results of effects of pH, protein concentration and contact time of the adsorption reveal that the morphology of adsorbed protein A is significantly affected by pH and adsorbed surface concentration, in which the pH away from the isoelectric point (IEP) of protein A could produce electrical repulsion to change the protein conformation, while the high adsorbed surface protein volume results in molecular networks. Protein A can form an adsorbed protein film on PDMS with a maximum volume of 2.45 x 10 -3 μm 3 . This work enhances our fundamental understanding of protein A adsorption on PDMS, a frequently used substrate component in miniaturized immunoassay devices.

  11. Competitive Adsorption of Plasma Proteins on Polysaccharide-Modified Silicon Surfaces

    National Research Council Canada - National Science Library

    Ombelli, Michela; Costello, Lauren B; Meng, Qing C; Composto, Russell J; Eckmann, David M

    2005-01-01

    .... Competitive protein adsorption plays a key role in the hemocompatibility of the surface. The synthesis of nonfouling surfaces is therefore one of the major prerequisites for devices for biomedical applications...

  12. Honey bee protein atlas at organ-level resolution.

    Science.gov (United States)

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed.

  13. Serum transferrin levels in children with protein-energy malnutrition

    Directory of Open Access Journals (Sweden)

    Selime Aydoğdu

    2013-03-01

    Full Text Available Objective: Although the diagnosis of patients with severemalnutrition is easy, it is very difficult to recognize patientswith mild and moderate malnutrition. A variety of methodsattempts to develop for early diagnosis of these cases.In this study, we evaluated the serum transferrin and albuminlevels in children with mild, moderate and severeprotein-energy malnutrition (PEM.Materials and methods: Children admitted to our policlinic,aged between 3 and 25 months, 45 subjects withPEM and 39 healthy subjects (control group were evaluated.According to the Gomez, Waterlow and Kanawatisubjects with PEM were divided in 3 subgroups mild,moderate and severe PEM. Anthropometric measurementsand biochemical results of 4 groups were compared.Results: For albumin levels in mild to moderate PEMgroups, 37.7% sensitivity, and 28.5% specificity, positivepredictive value 54%; negative predictive value 16.6%was found. For severe PEM sensitivity, specificity, positivepredictive value and negative predictive value were71%, 62.5%, 45%, and 83.3% respectively.With respect to the levels of transferrin, a significant differencewas found between mild PEM-control and moderatePEM-control groups (p0.05.Conclusion: Our study results showed that albumin isa weak indicator in mild-moderate PEM. In these cases,serum transferrin level reduces before decreasing of albuminlevel, thus it may be an early sensitive finding thatcan be used as a sensitive parameter in the diagnosis ofearly stages of malnutrition.Key words: Protein energy malnutrition, children, albumin,transferrin

  14. Photoswitchable method for the ordered attachment of proteins to surfaces

    Science.gov (United States)

    Camarero, Julio A.; De Yoreo, James J.; Kwon, Youngeun

    2010-04-20

    Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

  15. Modulating surface rheology by electrostatic protein/polysaccharide interactions

    NARCIS (Netherlands)

    Ganzevles, R.A.; Zinoviadou, K.; Vliet, van T.; Cohen Stuart, M.A.; Jongh, de H.H.J.

    2006-01-01

    There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/

  16. Photoswitchable method for the ordered attachment of proteins to surfaces

    Science.gov (United States)

    Camarero, Julio A [Livermore, CA; DeYoreo, James J [Clayton, CA; Kwon, Youngeun [Livermore, CA

    2011-07-05

    Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

  17. Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.

    Directory of Open Access Journals (Sweden)

    Taku Fukuzawa

    Full Text Available Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+ and Ig-Hepta(-/- mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i balanced synthesis of surfactant lipids and proteins and (ii surfactant secretion, and (iii a stimulating effect on recycling (uptake in response to elevated levels of Sp-D in alveolar space.

  18. Surface-Level Diversity and Decision-Making in Groups: When Does Deep-Level Similarity Help?

    OpenAIRE

    2006-01-01

    Abstract We examined how surface-level diversity (based on race) and deep-level similarities influenced three-person decision-making groups on a hidden-profile task. Surface-level homogeneous groups perceived their information to be less unique and spent less time on the task than surface-level diverse groups. When the groups were given the opportunity to learn about their deep-level similarities prior to t...

  19. the effect of dietary energy and protein levels on the composition

    African Journals Online (AJOL)

    Zannel

    Keywords: Breeding ostriches, nutrition, energy, protein, amino acids, egg ... Yolk is an important nutritional component of the avian egg because .... 3 (energy) x 3 (protein) factorial design with energy and protein levels featuring as main factors. ... No significant interactions were observed between energy and protein levels.

  20. Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

    International Nuclear Information System (INIS)

    Colledge, Danielle; Soppe, Sally; Yuen, Lilly; Selleck, Lucy; Walsh, Renae; Locarnini, Stephen; Warner, Nadia

    2017-01-01

    Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion. Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.

  1. Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Colledge, Danielle; Soppe, Sally; Yuen, Lilly; Selleck, Lucy; Walsh, Renae; Locarnini, Stephen, E-mail: stephen.locarnini@mh.org.au; Warner, Nadia

    2017-01-15

    Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion. Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.

  2. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

    Science.gov (United States)

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

    2014-06-01

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

  3. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Huihui; Qian, Bin; Zhang, Wei [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); Lan, Minbo, E-mail: minbolan@ecust.edu.cn [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2016-02-15

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU–PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU–PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU–PVP (6.0 h) film reduced greatly to 0.08 μg/cm{sup 2}, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  4. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    International Nuclear Information System (INIS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-01-01

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU–PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU–PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU–PVP (6.0 h) film reduced greatly to 0.08 μg/cm"2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  5. [Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

    Science.gov (United States)

    Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

    2017-10-01

    In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

  6. Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available Streptococcus suis serotype 2 (SS2 is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.

  7. Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

    International Nuclear Information System (INIS)

    Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

    2016-01-01

    Graphical abstract: - Highlights: • Dense package of poly(acrylamide) on poly(caprolactone) surface was achieved by surface-initiated atom transfer radical polymerization. • Poly(acrylamide) grafted surface exhibited high protein retention ability. • Loaded protein was resistant to detachment and maintained its structure without denaturation. - Abstract: To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

  8. Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

    Science.gov (United States)

    Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

    2016-12-01

    Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

  9. Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein.

    Directory of Open Access Journals (Sweden)

    Akinobu Kajikawa

    Full Text Available Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER from human immunodeficiency virus type 1 (HIV-1 within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

  10. SURF'S UP! – Protein classification by surface comparisons

    Indian Academy of Sciences (India)

    2006-12-12

    Dec 12, 2006 ... Large-scale genome sequencing and structural genomics projects generate numerous sequences and structures for 'hypothetical' proteins without functional characterizations. Detection of homology to experimentally characterized proteins can provide functional clues, but the accuracy of homology-based ...

  11. The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses

    NARCIS (Netherlands)

    K. Overweg (Karin); A. Kerr; M. Sluijter (Marcel); M.H. Jackson; T.J. Mitchell; A.P. de Jong; R. de Groot (Ronald); P.W.M. Hermans (Peter)

    2000-01-01

    textabstractSurface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune

  12. Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex

    DEFF Research Database (Denmark)

    Wichern, Franziska; Jensen, Majbrit M; Christensen, Ditte Z

    2017-01-01

    The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater...... degree in the frontal cortex (FC) compared with the hippocampus (HIP). We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the FC at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during...... a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal...

  13. In-cell thermodynamics and a new role for protein surfaces.

    Science.gov (United States)

    Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

    2016-02-16

    There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

  14. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...

  15. Maximum surface level and temperature histories for Hanford waste tanks

    International Nuclear Information System (INIS)

    Flanagan, B.D.; Ha, N.D.; Huisingh, J.S.

    1994-01-01

    Radioactive defense waste resulting from the chemical processing of spent nuclear fuel has been accumulating at the Hanford Site since 1944. This waste is stored in underground waste-storage tanks. The Hanford Site Tank Farm Facilities Interim Safety Basis (ISB) provides a ready reference to the safety envelope for applicable tank farm facilities and installations. During preparation of the ISB, tank structural integrity concerns were identified as a key element in defining the safety envelope. These concerns, along with several deficiencies in the technical bases associated with the structural integrity issues and the corresponding operational limits/controls specified for conduct of normal tank farm operations are documented in the ISB. Consequently, a plan was initiated to upgrade the safety envelope technical bases by conducting Accelerated Safety Analyses-Phase 1 (ASA-Phase 1) sensitivity studies and additional structural evaluations. The purpose of this report is to facilitate the ASA-Phase 1 studies and future analyses of the single-shell tanks (SSTs) and double-shell tanks (DSTs) by compiling a quantitative summary of some of the past operating conditions the tanks have experienced during their existence. This report documents the available summaries of recorded maximum surface levels and maximum waste temperatures and references other sources for more specific data

  16. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    International Nuclear Information System (INIS)

    Wise, K.S.; Kim, M.F.

    1987-01-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125 I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [ 35 S] methionine, 14 C-amino acids, or [ 3 H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11

  17. Structural determinants for protein adsorption/non-adsorption to silica surface

    International Nuclear Information System (INIS)

    Mathe, Christelle; Devineau, Stephanie; Aude, Jean-Christophe; Lagniel, Gilles; Chedin, Stephane; Legros, Veronique; Mathon, Marie-Helene; Renault, Jean-Philippe; Pin, Serge; Boulard, Yves; Labarre, Jean

    2013-01-01

    The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nano-technology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many p-p interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption. (authors)

  18. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    Energy Technology Data Exchange (ETDEWEB)

    Wise K.S.; Kim, M.F.

    1987-12-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

  19. Structural determinants for protein adsorption/non-adsorption to silica surface.

    Directory of Open Access Journals (Sweden)

    Christelle Mathé

    Full Text Available The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.

  20. Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.

    Science.gov (United States)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    2014-06-01

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Interactive effect of dietary protein level and zilpaterol hydrochloride ...

    African Journals Online (AJOL)

    p2492989

    feedlot performance and meat quality of steers ... result of decreased protein degradation and increased protein synthesis (Fiems, ... sheep as well as data from some BAA trials provide evidence to associate ... The perception is that meat is now ... Table 1 Dietary treatments of steers with a mean live weight of 278 ± 20 kg ...

  2. Effect of graded levels and sources of protein on scrotal ...

    African Journals Online (AJOL)

    Iso caloric rations (10.50 MJ/kg DM ME) were formulated using non-conventional protein source (maize offal and dry layer litter) to contain 12.11% CP, 14.96% CP, and 17.94% CP and fed to groups A, B and C respectively. Another ration was formulated using conventional protein source (maize, wheat bran, groundnut ...

  3. Exploring Sequence Characteristics Related to High- Level Production of Secreted Proteins in Aspergillus niger

    NARCIS (Netherlands)

    Van den Berg, B.A.; Reinders, M.J.T.; Hulsman, M.; Wu, L.; Pel, H.J.; Roubos, J.A.; De Ridder, D.

    2012-01-01

    Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large

  4. Selective cell-surface labeling of the molecular motor protein prestin

    International Nuclear Information System (INIS)

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    2011-01-01

    Highlights: → Trafficking to the plasma membrane is required for prestin function. → Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. → BAP-prestin can be metabolically labeled with biotin in HEK293 cells. → Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. → The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

  5. Probing protein surface with a solvent mimetic carbene coupled to detection by mass spectrometry.

    Science.gov (United States)

    Gómez, Gabriela E; Mundo, Mariana R; Craig, Patricio O; Delfino, José M

    2012-01-01

    Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH(2)). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites. © American Society for Mass Spectrometry, 2011

  6. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    International Nuclear Information System (INIS)

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-01-01

    Highlights: ► APPL1 regulates the protein level of EGFR in response to EGF stimulation. ► Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. ► Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  7. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  8. Characterization of Silk Fibroin Modified Surface: A Proteomic View of Cellular Response Proteins Induced by Biomaterials

    Directory of Open Access Journals (Sweden)

    Ming-Hui Yang

    2014-01-01

    Full Text Available The purpose of this study was to develop the pathway of silk fibroin (SF biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.

  9. Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

    Directory of Open Access Journals (Sweden)

    Yosef Y Kuttner

    2013-04-01

    Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

  10. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    International Nuclear Information System (INIS)

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-01-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [ 35 S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [ 3 H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125 I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [ 3 H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

  11. Experimental study on source efficiencies for estimating surface contamination level

    International Nuclear Information System (INIS)

    Ichiji, Takeshi; Ogino, Haruyuki

    2008-01-01

    Source efficiency was measured experimentally for various materials, such as metals, nonmetals, flooring materials, sheet materials and other materials, contaminated by alpha and beta emitter radioactive nuclides. Five nuclides, 147 Pm, 60 Co, 137 Cs, 204 Tl and 90 Sr- 90 Y, were used as the beta emitters, and one nuclide 241 Am was used as the alpha emitter. The test samples were prepared by placing drops of the radioactive standardized solutions uniformly on the various materials using an automatic quantitative dispenser system from Musashi Engineering, Inc. After placing drops of the radioactive standardized solutions, the test materials were allowed to dry for more than 12 hours in a draft chamber with a hood. The radioactivity of each test material was about 30 Bq. Beta rays or alpha rays from the test materials were measured with a 2-pi gas flow proportional counter from Aloka Co., Ltd. The source efficiencies of the metals, nonmetals and sheet materials were higher than 0.5 in the case of contamination by the 137 Cs, 204 Tl and 90 Sr- 90 Y radioactive standardized solutions, higher than 0.4 in the case of contamination by the 60 Co radioactive standardized solution, and higher than 0.25 in the case of contamination by the alpha emitter the 241 Am radioactive standardized solution. These values were higher than those given in Japanese Industrial Standards (JIS) documents. In contrast, the source efficiencies of some permeable materials were lower than those given in JIS documents, because source efficiency varies depending on whether the materials or radioactive sources are wet or dry. This study provides basic data on source efficiency, which is useful for estimating the surface contamination level of materials. (author)

  12. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taek [Research Institute for Basic Science, Sogang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Chung, Yong-Ho; Yoon, Jinho [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of); Min, Junhong [School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Choi, Jeong-Woo, E-mail: jwchoi@sogang.ac.kr [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of)

    2014-11-30

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

  13. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    International Nuclear Information System (INIS)

    Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

    2014-01-01

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development

  14. Non-interacting surface solvation and dynamics in protein-protein interactions

    NARCIS (Netherlands)

    Visscher, Koen M.; Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

    2015-01-01

    Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo

  15. DARC: Mapping Surface Topography by Ray-Casting for Effective Virtual Screening at Protein Interaction Sites.

    Science.gov (United States)

    Gowthaman, Ragul; Miller, Sven A; Rogers, Steven; Khowsathit, Jittasak; Lan, Lan; Bai, Nan; Johnson, David K; Liu, Chunjing; Xu, Liang; Anbanandam, Asokan; Aubé, Jeffrey; Roy, Anuradha; Karanicolas, John

    2016-05-12

    Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 μM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions.

  16. Effects of salts on protein-surface interactions: applications for column chromatography.

    Science.gov (United States)

    Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

    2007-07-01

    Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

  17. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  18. Sensing surface mechanical deformation using active probes driven by motor proteins

    Science.gov (United States)

    Inoue, Daisuke; Nitta, Takahiro; Kabir, Arif Md. Rashedul; Sada, Kazuki; Gong, Jian Ping; Konagaya, Akihiko; Kakugo, Akira

    2016-01-01

    Studying mechanical deformation at the surface of soft materials has been challenging due to the difficulty in separating surface deformation from the bulk elasticity of the materials. Here, we introduce a new approach for studying the surface mechanical deformation of a soft material by utilizing a large number of self-propelled microprobes driven by motor proteins on the surface of the material. Information about the surface mechanical deformation of the soft material is obtained through changes in mobility of the microprobes wandering across the surface of the soft material. The active microprobes respond to mechanical deformation of the surface and readily change their velocity and direction depending on the extent and mode of surface deformation. This highly parallel and reliable method of sensing mechanical deformation at the surface of soft materials is expected to find applications that explore surface mechanics of soft materials and consequently would greatly benefit the surface science. PMID:27694937

  19. Characterization of the Eimeria maxima sporozoite surface protein IMP1

    Science.gov (United States)

    The purpose of this study was to characterize Eimeria maxima immunoprotective protein IMP1 that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transc...

  20. 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.

  1. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface

    KAUST Repository

    Capriotti, Anna Laura

    2011-07-02

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids. © 2011 Springer-Verlag.

  2. Protein conformational transitions at the liquid-gas interface as studied by dilational surface rheology.

    Science.gov (United States)

    Noskov, Boris A

    2014-04-01

    Experimental results on the dynamic dilational surface elasticity of protein solutions are analyzed and compared. Short reviews of the protein behavior at the liquid-gas interface and the dilational surface rheology precede the main sections of this work. The kinetic dependencies of the surface elasticity differ strongly for the solutions of globular and non-globular proteins. In the latter case these dependencies are similar to those for solutions of non-ionic amphiphilic polymers and have local maxima corresponding to the formation of the distal region of the surface layer (type I). In the former case the dynamic surface elasticity is much higher (>60 mN/m) and the kinetic dependencies are monotonical and similar to the data for aqueous dispersions of solid nanoparticles (type II). The addition of strong denaturants to solutions of bovine serum albumin and β-lactoglobulin results in an abrupt transition from the type II to type I dependencies if the denaturant concentration exceeds a certain critical value. These results give a strong argument in favor of the preservation of the protein globular structure in the course of adsorption without any denaturants. The addition of cationic surfactants also can lead to the non-monotonical kinetic dependencies of the dynamic surface elasticity indicating destruction of the protein tertiary and secondary structures. The addition of anionic surfactants gives similar results only for the protein solutions of high ionic strength. The influence of cationic surfactants on the local maxima of the kinetic dependencies of the dynamic surface elasticity for solutions of a non-globular protein (β-casein) differs from the influence of anionic surfactants due to the heterogeneity of the charge distribution along the protein chain. In this case one can use small admixtures of ionic surfactants as probes of the adsorption mechanism. The effect of polyelectrolytes on the kinetic dependencies of the dynamic surface elasticity of protein

  3. Altering protein surface charge with chemical modification modulates protein–gold nanoparticle aggregation

    International Nuclear Information System (INIS)

    Jamison, Jennifer A.; Bryant, Erika L.; Kadali, Shyam B.; Wong, Michael S.; Colvin, Vicki L.; Matthews, Kathleen S.; Calabretta, Michelle K.

    2011-01-01

    Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein–AuNP assembly or influence the formation of the protein “corona,” modification of the protein surface as a mechanism to modulate protein–AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein–AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein–NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein–AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle–protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle–protein corona compositions.

  4. Protein resistance of surfaces modified with oligo(ethylene glycol) aryl diazonium derivatives.

    Science.gov (United States)

    Fairman, Callie; Ginges, Joshua Z; Lowe, Stuart B; Gooding, J Justin

    2013-07-22

    Anti-fouling surfaces are of great importance for reducing background interference in biosensor signals. Oligo(ethylene glycol) (OEG) moieties are commonly used to confer protein resistance on gold, silicon and carbon surfaces. Herein, we report the modification of surfaces using electrochemical deposition of OEG aryl diazonium salts. Using electrochemical and contact angle measurements, the ligand packing density is found to be loose, which supports the findings of the fluorescent protein labelling that aryl diazonium OEGs confer resistance to nonspecific adsorption of proteins albeit lower than alkane thiol-terminated OEGs. In addition to protein resistance, aryl diazonium attachment chemistry results in stable modification. In common with OEG species on gold electrodes, OEGs with distal hydroxyl moieties do confer superior protein resistance to those with a distal methoxy group. This is especially the case for longer derivatives where superior coiling of the OEG chains is possible. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  6. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  7. Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Madsen, Søren M; Glenting, Jacob

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...... of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics....

  8. Hybrid surface platform for the simultaneous detection of proteins and DNA using a surface plasmon resonance (SPR) imaging sensor

    Czech Academy of Sciences Publication Activity Database

    Homola, Jiří; Piliarik, Marek; Ladd, J.; Taylor, A.; Shaoyi, J.

    2008-01-01

    Roč. 80, č. 11 (2008), s. 4231-4236 ISSN 0003-2700 R&D Projects: GA AV ČR KAN200670701 Institutional research plan: CEZ:AV0Z20670512 Keywords : Surface plasmon resonance imaging * DNA-directed immobilization * protein array Subject RIV: JB - Sensors, Measurment, Regulation Impact factor: 5.712, year: 2008

  9. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    2010-07-01

    Full Text Available Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  10. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Science.gov (United States)

    Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

    2010-07-26

    Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  11. A Molecular-Level Account of the Antigenic Hantaviral Surface

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-05-01

    Full Text Available Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV, a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

  12. Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

    Directory of Open Access Journals (Sweden)

    Wouter S P Jong

    Full Text Available Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

  13. Zinc finger protein 521 overexpression increased transcript levels of ...

    Indian Academy of Sciences (India)

    2016-02-12

    Feb 12, 2016 ... Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human .... Membranes were blocked for 1 h with 10% skim milk and ..... fat-like development of white fat and thermogenesis.

  14. Serum total proteins and creatinine levels in experimental gambian ...

    African Journals Online (AJOL)

    Attempt was therefore made to evaluate the effect of two strains of Trypanosoma brucei gambiense on total proteins and other serum biochemical parameters using vervet monkeys as a model. The outcome of both strains in vervet monkeys was traumatic as the monkeys died from infection 12 – 15 weeks post infection while ...

  15. Plasma protein carbonyl levels and breast cancer risk

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Terry, M. B.; Gammon, M. D.; Agrawal, M.; Zhang, F. F.; Ferris, J.S.; Teitelbaum, S. L.; Eng, S. M.; Gaudet, M. M.; Neugut, A. I.; Santella, R. M.

    2007-01-01

    Roč. 11, č. 5 (2007), s. 1138-1148 ISSN 1582-1838 Institutional research plan: CEZ:AV0Z50390512 Keywords : oxidative stress * protein carbonyl * breast cancer Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 6.807, year: 2007

  16. Serum total protein, albumin and globulin levels in Trypanosoma ...

    African Journals Online (AJOL)

    The effect of orally administered Scoparia dulcis on Trypanosoma brucei-induced changes in serum total protein, albumin and globulin were investigated in rabbits over a period of twenty eight days. Results obtained show that infection resulted in hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. However ...

  17. Adsorption of plasma proteins : adsorption behaviour on apolar surfaces and effect on colloid stability

    NARCIS (Netherlands)

    van der Scheer, Albert

    1978-01-01

    In this thesis the adsorption of some plasma proteins (human albumin (HSA) and fibrinogen (HFb)) on non polar surfaces is studied, together with the influence of these proteins on the stability of polystyrene latices. The aim of these investigations is a better understanding of the processes

  18. Nitrate as a probe of cytochrome c surface : crystallographic identification of crucial "hot spots" for protein-protein recognition

    NARCIS (Netherlands)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive

  19. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    Science.gov (United States)

    Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

    2013-11-01

    The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

  20. Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi, E-mail: takeuchi@gold.kobe-u.ac.jp

    2016-08-15

    Highlights: • Three hydrophilic crosslinked polymers were examined for protein adsorption. • All polymers showed low nonspecific adsorption of negatively charged proteins. • Poly(MMPC) showed the lowest adsorption for positively charged proteins. • Poly(MMPC) is able to reduce nonspecific adsorption of a wide range of proteins. - Abstract: We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N′-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6′-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

  1. 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

  2. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

    Science.gov (United States)

    Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

  3. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

    Directory of Open Access Journals (Sweden)

    Manasi Balachandran

    Full Text Available Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A gene and binds to immunoglobulin (Ig. The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

  4. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

    Science.gov (United States)

    Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

    2017-01-01

    Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

  5. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    Science.gov (United States)

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-02

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

  6. Influence of surface features of hydroxyapatite on the adsorption of proteins relevant to bone regeneration.

    Science.gov (United States)

    Fernández-Montes Moraleda, Belén; San Román, Julio; Rodríguez-Lorenzo, Luís M

    2013-08-01

    Protein-surface interaction may determine the success or failure of an implanted device. Not much attention have been paid to the specific surface parametes of hydroxyapatite (OHAp) that modulates and determines the formation and potential activity of the layer of proteins that is first formed when the material get in contact with the host tissue. the influence of specific surface area (SSA), crystallite size (CS) and particle size (PS) of OHAp on the adsorption of proteins relevant for bone regeneration is evaluated in this article. OHAp have been prepared by a wet chemical reaction of Ca(OH)2 with H3PO4. One set of reactions included poly acrylic acid in the reactant solution to modify the properties of the powder. Fibrinogen (Fg) Fraction I, type I: from Human plasma, (67% Protein), and Fibronectin (Fn) from Human plasma were selected to perform the adsorption experiments. The analysis of protein adsorption was carried out by UV/Vis spectrometry. A lower SSA and a different aspect ratio are obtained when the acrylic acid is included in the reaction badge. The deconvolution of the amide I band on the Raman spectra of free and adsorbed proteins reveals that the interaction apatite-protein happens through the carboxylate groups of the proteins. The combined analysis of CS, SSA and PS should be considered on the design of OHAp materials intended to interact with proteins. Copyright © 2013 Wiley Periodicals, Inc.

  7. Evaluation of the Levels of phthalate Ester Plasticizers in Surface ...

    African Journals Online (AJOL)

    ADOWIE PERE

    Gas chromatography (GC) coupled with mass ... Key words: phthalates, acid esters, plasticizers, Ethiope River, surface water, pollution ... waste and emissions arising from burning of refuse ... deepest inland waterways in Africa, is known for its ..... carbon nitride nanocomposites for the solid-phase extraction of phthalate ...

  8. Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index

    DEFF Research Database (Denmark)

    Wang, Ping; Holst, Claus; Astrup, Arne

    2012-01-01

    ) blood biomarkers of dietary protein and GI levels during the weight-maintenance phase. Blood samples were collected at baseline, after 8 weeks of low-energy diet-induced weight loss and after a 6-month dietary intervention period from female continued weight losers (n 48) and weight regainers (n 48......), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some...

  9. Gold nanoparticles: role of size and surface chemistry on blood protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Benetti, F., E-mail: filippo.benetti@unitn.it; Fedel, M. [BIOtech Research Centre (Italy); Minati, L.; Speranza, G. [Fondazione Bruno Kessler (Italy); Migliaresi, C. [BIOtech Research Centre (Italy)

    2013-06-15

    Material interaction with blood proteins is a critical issue, since it could influence the biological processes taking place in the body following implantation/injection. This is particularly important in the case of nanoparticles, where innovative properties, such as size and high surface to volume ratio can lead to a behavioral change with respect to bulk macroscopic materials and could be responsible for a potential risk for human health. The aim of this work was to compare gold nanoparticles (AuNP) and planar surfaces to study the role of surface curvature moving from the macro- to the nano-size in the process of blood protein adsorption. In the course of the study, different protocols were tested to optimize the analysis of protein adsorption on gold nanoparticles. AuNP with different size (10, 60 and 200 nm diameter) and surface coatings (citrate and polyethylene glycol) were carefully characterized. The stabilizing action of blood proteins adsorbed on AuNP was studied measuring the variation of size and solubility of the nanoparticles following incubation with single protein solutions (human serum albumin and fibrinogen) and whole blood plasma. In addition, we developed a method to elute proteins from AuNP to study the propensity of gold materials to adsorb plasma proteins in function of dimensional characteristics and surface chemistry. We showed a different efficacy of the various eluting media tested, proving that even the most aggressive agent cannot provide a complete detachment of the protein corona. Enhanced protein adsorption was evidenced on AuNP if compared to gold laminae (bare and PEGylated) used as macroscopic control, probably due to the superior AuNP surface reactivity.

  10. Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

    Science.gov (United States)

    Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

    2008-08-25

    Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

  11. Identification of variant-specific surface proteins in Giardia muris trophozoites.

    Science.gov (United States)

    Ropolo, Andrea S; Saura, Alicia; Carranza, Pedro G; Lujan, Hugo D

    2005-08-01

    Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

  12. Identification of Variant-Specific Surface Proteins in Giardia muris Trophozoites

    OpenAIRE

    Ropolo, Andrea S.; Saura, Alicia; Carranza, Pedro G.; Lujan, Hugo D.

    2005-01-01

    Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

  13. Characterizing and modeling protein-surface interactions in lab-on-chip devices

    Science.gov (United States)

    Katira, Parag

    Protein adsorption on surfaces determines the response of other biological species present in the surrounding solution. This phenomenon plays a major role in the design of biomedical and biotechnological devices. While specific protein adsorption is essential for device function, non-specific protein adsorption leads to the loss of device function. For example, non-specific protein adsorption on bioimplants triggers foreign body response, in biosensors it leads to reduced signal to noise ratios, and in hybrid bionanodevices it results in the loss of confinement and directionality of molecular shuttles. Novel surface coatings are being developed to reduce or completely prevent the non-specific adsorption of proteins to surfaces. A novel quantification technique for extremely low protein coverage on surfaces has been developed. This technique utilizes measurement of the landing rate of microtubule filaments on kinesin proteins adsorbed on a surface to determine the kinesin density. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin-microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, a random sequential adsorption model is presented for protein adsorption to PEO-coated surfaces. The derived analytical expressions accurately predict the observed experimental results from various research groups, suggesting that PEO chains act as almost perfect steric barriers to protein adsorption. These expressions can be used to predict the performance of a variety of systems towards resisting protein adsorption and can help in the design of better non-fouling surface coatings. Finally, in biosensing systems, target analytes are captured and concentrated on specifically adsorbed proteins for detection. Non-specific adsorption of proteins results in the loss of signal, and an increase in the background. The use of nanoscale transducers as

  14. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  15. Microscopic Investigation of Reversible Nanoscale Surface Size Dependent Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Michael A. Carpenter

    2009-05-01

    Full Text Available Aβ1-40 coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aβ (a hallmark of Alzheimer’s disease. We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aβ aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.

  16. STS Observations of Landau Levels at Graphite Surfaces

    OpenAIRE

    Matsui, T.; Kambara, H.; Niimi, Y.; Tagami, K.; Tsukada, M.; Fukuyama, Hiroshi

    2004-01-01

    Scanning tunneling spectroscopy measurements were made on surfaces of two different kinds of graphite samples, Kish graphite and highly oriented pyrolytic graphite (HOPG), at very low temperatures and in high magnetic fields. We observed a series of peaks in the tunnel spectra, which grow with increasing field, both at positive and negative bias voltages. These are associated with Landau quantization of the quasi two-dimensional electrons and holes in graphite in magnetic fields perpendicular...

  17. CHEMICAL REACTIONS ON ADSORBING SURFACE: KINETIC LEVEL OF DESCRIPTION

    Directory of Open Access Journals (Sweden)

    P.P.Kostrobii

    2003-01-01

    Full Text Available Based on the effective Hubbard model we suggest a statistical description of reaction-diffusion processes for bimolecular chemical reactions of gas particles adsorbed on the metallic surface. The system of transport equations for description of particles diffusion as well as reactions is obtained. We carry out the analysis of the contributions of all physical processes to the formation of diffusion coefficients and chemical reactions constants.

  18. Effect of Dietary Protein Level on the Expression of Proteins in the Gastrointestinal Tract of Young Pigs.

    Science.gov (United States)

    Ma, Xianyong; Tian, Zhimei; Deng, Dun; Cui, Yiyan; Qiu, Yueqin

    2018-05-02

    The objective of this research is to investigate the effect of protein level on proteins expression in the gastrointestinal tract of young pigs. Eighteen piglets (Duroc × Landrace × Yorkshire) were weaned at 28 days of age and randomly assigned to three diets with 20%, 17%, and 14% CP level, and four essential amino acids, Lys, Met, Thr, and Trp, in three diets met the requirements of weaned piglets. The experimental period lasted 45 days. Compared with the control (20% CP level), the average daily feed intake, the average daily gain, and gain feed ratio of the 17% CP group did not decrease ( P > 0.05), but those of 14% CP group decreased ( P protein digestion and absorption, lipid or carbon digestion and absorption, etc. were up-regulated in 17% CP group, while most of them were down-regulated in 14% CP group. Amino acids metabolism of gastric, pancreatic secretion of duodenum or steroid hormone biosynthesis of jejunum was down-regulated in the 17% CP group, but the lipid metabolism was up-regulated in the 14% CP group. Six proteins were selected for identification by Western-blot, and their changes had the same trend as the proteomics results. The protein level decreased from 20% to 17%, the growth performance was not affected, while the nutrient digestion and absorption or the immune function were improved, which implied that 17% protein level maybe benefit for nutrients absorption of pigs.

  19. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    Science.gov (United States)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  20. InterProSurf: a web server for predicting interacting sites on protein surfaces

    Science.gov (United States)

    Negi, Surendra S.; Schein, Catherine H.; Oezguen, Numan; Power, Trevor D.; Braun, Werner

    2009-01-01

    Summary A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. PMID:17933856

  1. Genetic diversity of three surface protein genes in Plasmodium malariae from three Asian countries.

    Science.gov (United States)

    Srisutham, Suttipat; Saralamba, Naowarat; Sriprawat, Kanlaya; Mayxay, Mayfong; Smithuis, Frank; Nosten, Francois; Pukrittayakamee, Sasithon; Day, Nicholas P J; Dondorp, Arjen M; Imwong, Mallika

    2018-01-11

    Genetic diversity of the three important antigenic proteins, namely thrombospondin-related anonymous protein (TRAP), apical membrane antigen 1 (AMA1), and 6-cysteine protein (P48/45), all of which are found in various developmental stages of Plasmodium parasites is crucial for targeted vaccine development. While studies related to the genetic diversity of these proteins are available for Plasmodium falciparum and Plasmodium vivax, barely enough information exists regarding Plasmodium malariae. The present study aims to demonstrate the genetic variations existing among these three genes in P. malariae by analysing their diversity at nucleotide and protein levels. Three surface protein genes were isolated from 45 samples collected in Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4), using conventional polymerase chain reaction (PCR) assay. Then, the PCR products were sequenced and analysed using BioEdit, MEGA6, and DnaSP programs. The average pairwise nucleotide diversities (π) of P. malariae trap, ama1, and p48/45 were 0.00169, 0.00413, and 0.00029, respectively. The haplotype diversities (Hd) of P. malariae trap, ama1, and p48/45 were 0.919, 0.946, and 0.130, respectively. Most of the nucleotide substitutions were non-synonymous, which indicated that the genetic variations of these genes were maintained by positive diversifying selection, thus, suggesting their role as a potential target of protective immune response. Amino acid substitutions of P. malariae TRAP, AMA1, and P48/45 could be categorized to 17, 20, and 2 unique amino-acid variants, respectively. For further vaccine development, carboxyl terminal of P48/45 would be a good candidate according to conserved amino acid at low genetic diversity (π = 0.2-0.3). High mutational diversity was observed in P. malariae trap and ama1 as compared to p48/45 in P. malariae samples isolated from Thailand, Myanmar, and Lao PDR. Taken together, these results suggest that P48/45 might be a good vaccine

  2. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    International Nuclear Information System (INIS)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized ( 125 I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis

  3. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  4. Facile Photoimmobilization of Proteins onto Low-Binding PEG-Coated Polymer Surfaces

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Mikkelsen, Morten Bo Lindholm; Larsen, Niels Bent

    2014-01-01

    was verified for both enzymes and antibodies, and their presence on the surface was confirmed by X-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy. Conjugation of capture antibody onto the PEG coating was employed for a simplified ELISA protocol without the need for blocking uncoated...... surface areas, showing ng/mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features....

  5. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

    Directory of Open Access Journals (Sweden)

    Mariya Tarazanova

    2017-09-01

    Full Text Available Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities.

  6. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

    Science.gov (United States)

    Tarazanova, Mariya; Huppertz, Thom; Beerthuyzen, Marke; van Schalkwijk, Saskia; Janssen, Patrick; Wels, Michiel; Kok, Jan; Bachmann, Herwig

    2017-01-01

    Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities. PMID:28936202

  7. Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

    NARCIS (Netherlands)

    Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

    1995-01-01

    Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

  8. Protein arrangement on modified diamond-like carbon surfaces – An ARXPS study

    Energy Technology Data Exchange (ETDEWEB)

    Oosterbeek, Reece N., E-mail: reece.oosterbeek@auckland.ac.nz [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019 (New Zealand); Seal, Christopher K. [Light Metals Research Centre, The University of Auckland, Private Bag 92019 (New Zealand); Hyland, Margaret M. [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019 (New Zealand)

    2014-12-01

    Highlights: • DLC coatings were modified by Ar{sup +} ion sputtering and laser graphitisation. • The surface properties of the coatings were measured, and it was found that the above methods increased sp{sup 2} content and altered surface energy. • ARXPS was used to observe protein arrangement on the surface. • Polar CO/CN groups were seen to be segregated towards the interface, indicating they play an important role in bonding. • This segregation increased with increasing polar surface energy, indicating an increased net attraction between polar groups. - Abstract: Understanding the nature of the interface between a biomaterial implant and the biological fluid is an essential step towards creating improved implant materials. This study examined a diamond-like carbon coating biomaterial, the surface energy of which was modified by Ar{sup +} ion sputtering and laser graphitisation. The arrangement of proteins was analysed by angle resolved X-ray photoelectron spectroscopy, and the effects of the polar component of surface energy on this arrangement were observed. It was seen that polar groups (such as CN, CO) are more attracted to the coating surface due to the stronger polar interactions. This results in a segregation of these groups to the DLC–protein interface; at increasing takeoff angle (further from to DLC–protein interface) fewer of these polar groups are seen. Correspondingly, groups that interact mainly by dispersive forces (CC, CH) were found to increase in intensity as takeoff angle increased, indicating they are segregated away from the DLC–protein interface. The magnitude of the segregation was seen to increase with increasing polar surface energy, this was attributed to an increased net attraction between the solid surface and polar groups at higher polar surface energy (γ{sub S}{sup p})

  9. Protein arrangement on modified diamond-like carbon surfaces – An ARXPS study

    International Nuclear Information System (INIS)

    Oosterbeek, Reece N.; Seal, Christopher K.; Hyland, Margaret M.

    2014-01-01

    Highlights: • DLC coatings were modified by Ar + ion sputtering and laser graphitisation. • The surface properties of the coatings were measured, and it was found that the above methods increased sp 2 content and altered surface energy. • ARXPS was used to observe protein arrangement on the surface. • Polar CO/CN groups were seen to be segregated towards the interface, indicating they play an important role in bonding. • This segregation increased with increasing polar surface energy, indicating an increased net attraction between polar groups. - Abstract: Understanding the nature of the interface between a biomaterial implant and the biological fluid is an essential step towards creating improved implant materials. This study examined a diamond-like carbon coating biomaterial, the surface energy of which was modified by Ar + ion sputtering and laser graphitisation. The arrangement of proteins was analysed by angle resolved X-ray photoelectron spectroscopy, and the effects of the polar component of surface energy on this arrangement were observed. It was seen that polar groups (such as CN, CO) are more attracted to the coating surface due to the stronger polar interactions. This results in a segregation of these groups to the DLC–protein interface; at increasing takeoff angle (further from to DLC–protein interface) fewer of these polar groups are seen. Correspondingly, groups that interact mainly by dispersive forces (CC, CH) were found to increase in intensity as takeoff angle increased, indicating they are segregated away from the DLC–protein interface. The magnitude of the segregation was seen to increase with increasing polar surface energy, this was attributed to an increased net attraction between the solid surface and polar groups at higher polar surface energy (γ S p )

  10. Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

    Directory of Open Access Journals (Sweden)

    Lilyan Chan

    Full Text Available BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

  11. Structural dependence of the 5d-metal surface energies as deduced from surface core-level shift measurements

    International Nuclear Information System (INIS)

    Mrartensson, N.; Saalfeld, H.B.; Kuhlenbeck, H.; Neumann, M.

    1989-01-01

    Surface core-level shift measurements performed at the BESSY storage ring yield -0.41(2) eV for Os(0001) and 0.00(10) eV for Re(0001). An analysis of the surface shifts in the 5d transition series shows that the surface energy as a function of Z has a maximum at lower Z for the bcc phase than for the fcc-hcp phases, at W and between Re and Os, respectively

  12. 3D-SURFER: software for high-throughput protein surface comparison and analysis.

    Science.gov (United States)

    La, David; Esquivel-Rodríguez, Juan; Venkatraman, Vishwesh; Li, Bin; Sael, Lee; Ueng, Stephen; Ahrendt, Steven; Kihara, Daisuke

    2009-11-01

    We present 3D-SURFER, a web-based tool designed to facilitate high-throughput comparison and characterization of proteins based on their surface shape. As each protein is effectively represented by a vector of 3D Zernike descriptors, comparison times for a query protein against the entire PDB take, on an average, only a couple of seconds. The web interface has been designed to be as interactive as possible with displays showing animated protein rotations, CATH codes and structural alignments using the CE program. In addition, geometrically interesting local features of the protein surface, such as pockets that often correspond to ligand binding sites as well as protrusions and flat regions can also be identified and visualized. 3D-SURFER is a web application that can be freely accessed from: http://dragon.bio.purdue.edu/3d-surfer dkihara@purdue.edu Supplementary data are available at Bioinformatics online.

  13. Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface.

    Directory of Open Access Journals (Sweden)

    Ya-Ping Ko

    Full Text Available Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

  14. Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

    Directory of Open Access Journals (Sweden)

    Mickaël Desvaux

    2018-02-01

    Full Text Available The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria is formed of a cytoplasmic membrane (CM and a cell wall (CW. While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226, i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658, i.e., the lipoproteins. At the CW (GO: 0009275, cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed.

  15. GHRSST Level 4 DMI_OI Global Foundation Sea Surface Temperature Analysis (GDS version 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis by the Danish...

  16. GHRSST Level 4 MUR North America Regional Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced as a retrospective dataset at the JPL Physical...

  17. GHRSST Level 4 OSPO Global Nighttime Foundation Sea Surface Temperature Analysis (GDS version 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the Office of...

  18. GHRSST Level 4 AVHRR_AMSR_OI Global Blended Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) global Level 4 sea surface temperature analysis produced daily on a 0.25 degree grid at the NOAA...

  19. GHRSST Level 4 ODYSSEA Mediterranean Sea Regional Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at Ifremer/CERSAT...

  20. GHRSST Level 4 G1SST Global Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis by the JPL OurOcean...

  1. GHRSST Level 4 MW_OI Global Foundation Sea Surface Temperature analysis (GDS version 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) global Level 4 sea surface temperature analysis produced daily on a 0.25 degree grid at Remote Sensing...

  2. GHRSST Level 4 K10_SST Global 1 meter Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the Naval...

  3. GHRSST Level 4 EUR Mediterranean Sea Regional Foundation Sea Surface Temperature Analysis (GDS version 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily by Ifremer/CERSAT (France) using optimal...

  4. GHRSST Level 4 ODYSSEA Eastern Central Pacific Regional Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at Ifremer/CERSAT...

  5. GHRSST Level 4 ODYSSEA Global Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at Ifremer/CERSAT...

  6. GHRSST Level 4 OSPO Global Foundation Sea Surface Temperature Analysis (GDS version 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the Office of...

  7. GHRSST Level 4 GAMSSA Global Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the Australian Bureau...

  8. GHRSST Level 4 RAMSSA Australian Regional Foundation Sea Surface Temperature Analysis (GDS version 1)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the Australian Bureau...

  9. GHRSST Level 4 OSTIA Global Foundation Sea Surface Temperature Analysis (GDS versions 1 and 2)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A Group for High Resolution Sea Surface Temperature (GHRSST) Level 4 sea surface temperature analysis produced daily on an operational basis at the UK Met Office...

  10. Radioactivity levels in surface water of lakes around Izmir / Turkey

    International Nuclear Information System (INIS)

    Doyurum, S.; Turkozu, D. A.; Aslani, M. A. A.; Aytas, S.; Eral, M.; Kaygun, A. K.

    2006-01-01

    Radioactivity presents in surface continental waters is mainly due to the presence of radioactive elements in the earth's crust, other artificial radionuclides have appeared due to such human activities as nuclear power plants, nuclear weapons testing and manufacture and use of radioactive sources It is well known that natural radionuclides can be effective as tracers for the different processes controlling the distribution of elements among dissolved and particulate phases in aquatic systems. The detection of high radionuclide concentrations was proposed as a public health problem in several areas and consequently studies into the risks of radionuclides were started in the 2000s. Especially, these radioactive substances in groundwater are an unwanted and involuntary risk factor from natural sources, not artificial sources. These radioactive substances include uranium, radon found in uranium series, and other radioactive substances such as radium and gross alpha. Uranium present in rock, soil, and natural materials, and is found in small quantities in air, water, and food that people always contact. In this project, lake water samples were collected from three lakes around Izmir-Turkey. In surface lake water samples, pH, mV and conductivity values were measured and alkaline content was determined titrimetrically. The uranium concentrations in the lake water samples were measured using uranium analyzer. The radioactivity concentrations related to gross radium isotopes, gross-? and gross-? activities in the surface lake water were determined. The correlation among some parameters for water samples and concentrations of uranium, activity concentration of gross radium isotopes, gross alpha and gross beta radioactivity are also discussed

  11. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  12. FJ-2207 measuring instrument detection pipe surface a level of pollution method

    International Nuclear Information System (INIS)

    Wang Jiangong

    2010-01-01

    On the pipe surface contamination were detected α level of pollution is a frequently encountered dose-detection work. Because the pipeline surface arc, while the measuring probe for the plane, which for accurate measurement difficult. In this paper, on the FJ-2207-type pipe surface contamination measuring instrument measuring pollution levels in the α method was studied. Introduced the FJ-2207 measuring instrument detection pipe surface α pollution levels. Studied this measuring instrument on the same sources of surface, plane α level of radioactivity measured differences in the results obtained control of the apparatus when the direct measurement of the surface correction factor, and gives 32-216 specifications commonly used pipe direct measurement of the amendment factor. Convenient method, test results are reliable for the accurate measurement of pipe pollution levels in the surface of α as a reference and learning. (authors)

  13. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kang, C.W.; Tungsanga, K.; Walser, M.

    1986-04-01

    The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. /sup 14/C-KIC and /sup 3/H-leucine are injected orally. Six hours later the ratio, R, of /sup 14/C//sup 3/H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake.

  14. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

    International Nuclear Information System (INIS)

    Kang, C.W.; Tungsanga, K.; Walser, M.

    1986-01-01

    The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. 14 C-KIC and 3 H-leucine are injected orally. Six hours later the ratio, R, of 14 C/ 3 H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake

  15. 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.

  16. Surface finishing and levelling of thermomechanically hardened rolled steel

    International Nuclear Information System (INIS)

    Grosval'd, V.G.; Bashchenko, A.P.; Grishkov, A.I.; Gutnik, M.V.; Kanevskij, B.L.; Nikozov, A.I.; Sedov, N.D.; Prosin, K.A.; Safonov, L.I.

    1975-01-01

    The finishing of high-strength merchant shapes from alloy steel was tried out under industrial conditions with the equipment of metallurgical plants. After thermomechanical hardening in the production line of the rolling mill, 30KhGSN2A and 40Kh1NVA steel rounds 32 and 31 mm in diameter were straightened on a two-roller straightening machine designed by the All-Union Scientific Research Institute for Metallurgical Machinery (VNII Metmash). This made possible subsequent turning and grinding of the rods. The conditions of straightening, turning and grinding have been worked so as to obtain thermomechanically strengthened and ground rolled products approximating the gauged and ground metal in shape geometry and surface finish. It is shown that the labour-consuming operation of turning can be eliminated by reducing the machining pass of the rolled product, and this lowers the labour required for the finishing operations by 75%. After grinding with 40- and 25-grain abrasive wheels, high strength rolled shapes were obtained with a diameter of 30-0.20 mm and a surface finish of class 6-5 satisfying the technical specifications. (author)

  17. Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2008-07-01

    Full Text Available Abstract Background Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins. Results By assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states. Conclusion Our technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold is available at http://faculty.cs.tamu.edu/shsze/ssfold.

  18. Effects of dietary protein level on growth, health and physiological parameters in growing-furring mink

    DEFF Research Database (Denmark)

    Damgaard, Birthe Marie; Larsen, Peter F.; Clausen, Tove

    2012-01-01

    The aim of the study was to investigate the effects of the dietary protein level and the feeding strategy on growth, health and physiological blood and liver parameters in growing-furring male mink. Effects of dietary protein levels ranging from 22% of metabolizable energy (MEp) to experimental p...

  19. Effect of nonsteroidal antiinflammatory drugs on the C-reactive protein level in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Tarp, Simon; Bartels, Else M.; Bliddal, Henning

    2012-01-01

    To evaluate the effects of oral nonsteroidal antiinflammatory drugs (NSAIDs) on C-reactive protein (CRP) levels in rheumatoid arthritis (RA) patients, with a prespecified focus on the different NSAIDs.......To evaluate the effects of oral nonsteroidal antiinflammatory drugs (NSAIDs) on C-reactive protein (CRP) levels in rheumatoid arthritis (RA) patients, with a prespecified focus on the different NSAIDs....

  20. Effect of dietary protein and energy levels on the growth ...

    African Journals Online (AJOL)

    Dr. A.O. Ani

    While there were significant interactions (P< 0.05) between TBNO and enzyme levels on DM, ... ingredients like soya bean meal, groundnut meal, etc vis- à-vis their acute ... bacterial infection and sulfaquinoxaline drugs against coccidioses.

  1. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    Science.gov (United States)

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans. © 2015 by The Mycological Society of America.

  2. Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

    Science.gov (United States)

    Zhang, Cathy X. Y.; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

    2016-01-01

    ABSTRACT The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. IMPORTANCE L. monocytogenes is

  3. Respon Pertumbuhan Ayam Lokal Pedaging terhadap Suplementasi Protein Isolasi Biji-bijian (PIB dan Perbedaan Level Protein Ransum

    Directory of Open Access Journals (Sweden)

    M. Aman Yaman

    2009-10-01

    Full Text Available The response of local meat chicken growth to supplementation of isolated grain protein and the difference in ration protein level ABSTRACT. A research which aims to determine the response of local meat chicken growth of protein supplementation with Isolation Grains Protein (IGB and the difference in ration protein level has been conducted in the Laboratory of Experimental Farm, Animal Husbandry Department, Faculty of Agriculture, Syiah Kuala University-Darussalam, Banda Aceh for 90 days. This study used a completely randomized design factorial with 2 factors, consisting of factors namely male gender (JJ and female (JB and the ration is a combination of factors and levels IGB in the ration, ie: treatment A: 17% protein and 0.4% IGB; treatment B 19% protein and 0.6% IGB and treatment C 21% protein and 0.8% IGB. Each combination consisted of 4 replications and each replication consists of 5 chickens. Parameters observed in the study were weight gain, achievement of final weight, consumption, conversion and efficiency of ration. DOC used a derivative result of selection of local meat chicken which are in the process of selection. Data acquired and processed by ANOVA. The results showed that supplementation of IGB and ration protein level difference was significantly effect (P <0.01 on weight gain, final weight, rate of consumption, conversion efficiency of rations and rations, but there is no interaction effect between sex and ration factors . The highest weight gain obtained in the male local chicken achieved by feeding a ration B (93.23 grams, while the hen rations achieved by providing treatment C (63.86 grams / week. The highest final body weight of male chicken on treatment B (1491.5 gram/90 days and hens in treatment C (1061.5 gram/90 days. However, the highest ration consumption in both male and female local chickens obtained from the ration A. Feed conversion value and the best feed efficiency obtained in treatment B for the treatment of

  4. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    Science.gov (United States)

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  5. Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-01-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

  6. Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-04-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

  7. The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

    Science.gov (United States)

    Miranda-Ozuna, Jesús F T; Hernández-García, Mar S; Brieba, Luis G; Benítez-Cardoza, Claudia G; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2016-10-01

    Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Laser surface processing with controlled nitrogen-argon concentration levels for regulated surface life time

    Science.gov (United States)

    Obeidi, M. Ahmed; McCarthy, E.; Brabazon, D.

    2018-03-01

    Laser surface modification can be used to enhance the mechanical properties of a material, such as hardness, toughness, fatigue strength, and corrosion resistance. Surface nitriding is a widely used thermochemical method of surface modification, in which nitrogen is introduced into a metal or other material at an elevated temperature within a furnace. It is used on parts where there is a need for increased wear resistance, corrosion resistance, fatigue life, and hardness. Laser nitriding is a novel method of nitriding where the surface is heated locally by a laser, either in an atmosphere of nitrogen or with a jet of nitrogen delivered to the laser heated site. It combines the benefits of laser modification with those of nitriding. Recent work on high toughness tool steel samples has shown promising results due to the increased nitrogen gas impingement onto the laser heated region. Increased surface activity and nitrogen adsorption was achieved which resulted in a deeper and harder surface compared to conventional hardening methods. In this work, the effects of the laser power, pulse repetition frequency, and overlap percentage on laser surface treatment of 316 L SST steel samples with an argon-nitrogen jet will be presented. Resulting microstructure, phase type, microhardness, and wear resistance are presented.

  9. Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

    International Nuclear Information System (INIS)

    Ramanaviciene, A.; Kausaite-Minkstimiene, A.; Voronovic, J.; Ramanavicius, A.; Oztekin, Y.; Carac, G.; German, N.

    2011-01-01

    We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces. (author)

  10. Multidrug resistance-associated protein-1 (MRP1 genetic variants, MRP1 protein levels and severity of COPD

    Directory of Open Access Journals (Sweden)

    Rutgers Bea

    2010-05-01

    Full Text Available Abstract Background Multidrug resistance-associated protein-1 (MRP1 protects against oxidative stress and toxic compounds generated by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease (COPD. We have previously shown that single nucleotide polymorphisms (SNPs in MRP1 significantly associate with level of FEV1 in two independent population based cohorts. The aim of our study was to assess the associations of MRP1 SNPs with FEV1 level, MRP1 protein levels and inflammatory markers in bronchial biopsies and sputum of COPD patients. Methods Five SNPs (rs212093, rs4148382, rs504348, rs4781699, rs35621 in MRP1 were genotyped in 110 COPD patients. The effects of MRP1 SNPs were analyzed using linear regression models. Results One SNP, rs212093 was significantly associated with a higher FEV1 level and less airway wall inflammation. Another SNP, rs4148382 was significantly associated with a lower FEV1 level, higher number of inflammatory cells in induced sputum and with a higher MRP1 protein level in bronchial biopsies. Conclusions This is the first study linking MRP1 SNPs with lung function and inflammatory markers in COPD patients, suggesting a role of MRP1 SNPs in the severity of COPD in addition to their association with MRP1 protein level in bronchial biopsies.

  11. Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

    International Nuclear Information System (INIS)

    Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

    1976-01-01

    Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

  12. An Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

    Science.gov (United States)

    McClintock, Carlee S; Hettich, Robert L.

    2012-01-01

    Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins. PMID:23210708

  13. The ability of IgY to recognize surface proteins of Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Basri A. Gani

    2009-12-01

    Full Text Available Background: Streptococcus mutans are gram positive bacteria classified into viridians group, and have a role in pathogenesis of dental caries. It’s adhesion to the tooth surface is mediated by cell surface proteins, which interact with specific receptor located in tooth pellicle. Glucan binding protein, Glukosyltransferase, and antigen I/II are basic proteins of S. mutans, which have a role in initiating the interaction. A previous study showed that chicken’s IgY can interfere the interaction. Purpose: The objective of this study was to assess the ability of IgY in recognizing the surface molecule of Streptococcus mutans expressed by various serotypes (c, d, e, f and a strain derived from IPB, Bogor. Method: Western blot was used as a method to determine such capability. Result: The result showed that IgY has a potency to recognize antigen I/II, but not the other proteins on the cell surface of all bacteria tested. Conclusion: The ability of IgY to bind the surface protein, antigen I/II, indicates that this avian antibody could be used as a candidate for anti-adhesion in preventing dental caries.

  14. Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

    Science.gov (United States)

    Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

    2012-01-01

    The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

  15. In vitro investigation of protein adsorption and platelet adhesion on inorganic biomaterial surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Yan Huang [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Lue Xiaoying [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China)], E-mail: luxy@seu.edu.cn; Ma Jingwu [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Nan Huang [Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China)], E-mail: nhuang@263.com

    2008-11-15

    The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the ratio of adsorbed albumin (Alb) to IgG (R{sub A/I}) on DLC is larger than Ti, which coincide with the sequence of the ratio of interfacial tension between solid surface and Alb ({gamma}{sub S,Alb}) to interfacial tension between surface and IgG ({gamma}{sub S,IgG}) ({gamma}{sub S,Alb}/{gamma}{sub S,IgG}). The DLC film has a preferential adsorption for Alb. The results suggest that the ratio of {gamma}{sub S,Alb}/{gamma}{sub S,IgG} may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood compatibility of DLC seems to be better than Ti.

  16. 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.

  17. Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors

    Directory of Open Access Journals (Sweden)

    Rushikesh Sable

    2015-06-01

    Full Text Available Blocking protein-protein interactions (PPI using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

  18. Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors.

    Science.gov (United States)

    Sable, Rushikesh; Jois, Seetharama

    2015-06-23

    Blocking protein-protein interactions (PPI) using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

  19. A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Goda, Shuichiro; Koga, Tomoyuki; Yamashita, Kenichiro; Kuriura, Ryo; Ueda, Toshifumi

    2018-04-08

    In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

  20. Selective radiolabeling of cell surface proteins to a high specific activity

    International Nuclear Information System (INIS)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-01-01

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125 I-(hydroxyphenyl)propionyl groups via 125 I-sulfosuccinimidyl (hydroxyphenyl)propionate ( 125 II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[ 125 I]iodide labeling technique revealed that 125 I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125 I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane

  1. Analysis of direct immobilized recombinant protein G on a gold surface

    International Nuclear Information System (INIS)

    Kim, Hyunhee; Kang, Da-Yeon; Goh, Hyun-Jeong; Oh, Byung-Keun; Singh, Ravindra P.; Oh, Soo-Min; Choi, Jeong-Woo

    2008-01-01

    Abstact: For the immobilization of IgG, various techniques such as chemical linker, thiolated protein G methods, and fragmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoglobulin Fc-binding B-domain of protein G to contain two cysteine residues at its C-terminus by a genetic engineering technique. The resulting recombinant protein, RPGcys, retained IgG-binding activity in the same manner as native protein G. RPGcys was immobilized on a gold surface by strong affinity between thiol of cysteine and gold. The orientations of both IgG layers immobilized on the base recombinant protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IgG-binding activity of RPGcys on gold surface significantly increased in comparison to wild type of protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of highly oriented antibodies based on cysteine-modified protein G could be useful for the fabrication of immunosensor systems

  2. Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity.

    Science.gov (United States)

    Conde, José Miñones; Escobar, María del Mar Yust; Pedroche Jiménez, Justo J; Rodríguez, Francisco Millán; Rodríguez Patino, Juan M

    2005-10-05

    Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.

  3. Fast protein tertiary structure retrieval based on global surface shape similarity.

    Science.gov (United States)

    Sael, Lee; Li, Bin; La, David; Fang, Yi; Ramani, Karthik; Rustamov, Raif; Kihara, Daisuke

    2008-09-01

    Characterization and identification of similar tertiary structure of proteins provides rich information for investigating function and evolution. The importance of structure similarity searches is increasing as structure databases continue to expand, partly due to the structural genomics projects. A crucial drawback of conventional protein structure comparison methods, which compare structures by their main-chain orientation or the spatial arrangement of secondary structure, is that a database search is too slow to be done in real-time. Here we introduce a global surface shape representation by three-dimensional (3D) Zernike descriptors, which represent a protein structure compactly as a series expansion of 3D functions. With this simplified representation, the search speed against a few thousand structures takes less than a minute. To investigate the agreement between surface representation defined by 3D Zernike descriptor and conventional main-chain based representation, a benchmark was performed against a protein classification generated by the combinatorial extension algorithm. Despite the different representation, 3D Zernike descriptor retrieved proteins of the same conformation defined by combinatorial extension in 89.6% of the cases within the top five closest structures. The real-time protein structure search by 3D Zernike descriptor will open up new possibility of large-scale global and local protein surface shape comparison. 2008 Wiley-Liss, Inc.

  4. Synthesis of an endothelial cell mimicking surface containing thrombomodulin and endothelial protein C receptor

    Science.gov (United States)

    Kador, Karl Erich

    Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay

  5. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of α-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the

  6. Enhanced Hydrophilicity and Protein Adsorption of Titanium Surface by Sodium Bicarbonate Solution

    Directory of Open Access Journals (Sweden)

    Shengnan Jia

    2015-01-01

    Full Text Available The aim of this study was to investigate a novel and convenient method of chemical treatment to modify the hydrophilicity of titanium surfaces. Sand-blasted and acid-etched (SLA titanium surfaces and machined titanium surfaces were treated with sodium bicarbonate (NaHCO3 solution. The wetting behavior of both kinds of surfaces was measured by water contact angle (WCA test. The surface microstructure was assessed with scanning electron microscopy (SEM and three-dimensional (3D optical microscopy. The elemental compositions of the surfaces were analyzed by X-ray photoelectron spectroscopy (XPS. The protein adsorption analysis was performed with fibronectin. Results showed that, after 1 M NaHCO3 treatment, the hydrophilicity of both SLA and machined surfaces was enhanced. No significant microstructural change presented on titanium surfaces after NaHCO3 treatment. The deprotonation and ion exchange activities might cause the enhanced hydrophilicity of titanium surfaces. The increased protein adsorption of NaHCO3-treated SLA surfaces might indicate their improved tissue-integration in clinical use.

  7. Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

    Science.gov (United States)

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2018-02-10

    A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. GLUCOSE AND TOTAL PROTEIN LEVEL IN LABORATORY RATS UNDER CONDITIONS OF SHORT-TERM FASTING

    Directory of Open Access Journals (Sweden)

    Damir Suljević

    2013-09-01

    Full Text Available Glucose level (UV enzymatic method and total protein level (Biuret method were measured in the blood samples of the rats exposed to short-term starvation. We found a statistically significant increase in the glucose level in experimental animals during starvation, which is also evident in males and females in the experimental group (p <0.05, while decrease in the total protein level was not statistically significant. During starvation, more significant weight loss was observed in females compared to males.Key words: glucose, total protein, serum, Rattus

  9. Effect of dietary crude protein level on the performance and ...

    African Journals Online (AJOL)

    ویرایه

    2013-06-26

    Jun 26, 2013 ... kids who were 86 ± 3 days old with live weight of 9 ± 03 kg were used in a completely randomized design. ... matter intake (DMI) than the kids fed with 10.5, 12.8, .... black goats fed with 18% CP level with 20, 16 and 14%.

  10. Sublethal Effects of Diesel on Total Protein Levels and Cholesterol ...

    African Journals Online (AJOL)

    Michael Horsfall

    were handpicked at the Eagle Cement area of the New Calabar River and subjected to different levels ... groups when compared to the control in both the muscle and the viscera of the periwinkle. ... In Nigeria, oil industry operations are both offshore and onshore. ... This acts as a means of assessing the hazard or potential ...

  11. Protein arrangement on modified diamond-like carbon surfaces - An ARXPS study

    Science.gov (United States)

    Oosterbeek, Reece N.; Seal, Christopher K.; Hyland, Margaret M.

    2014-12-01

    Understanding the nature of the interface between a biomaterial implant and the biological fluid is an essential step towards creating improved implant materials. This study examined a diamond-like carbon coating biomaterial, the surface energy of which was modified by Ar+ ion sputtering and laser graphitisation. The arrangement of proteins was analysed by angle resolved X-ray photoelectron spectroscopy, and the effects of the polar component of surface energy on this arrangement were observed. It was seen that polar groups (such as CN, CO) are more attracted to the coating surface due to the stronger polar interactions. This results in a segregation of these groups to the DLC-protein interface; at increasing takeoff angle (further from to DLC-protein interface) fewer of these polar groups are seen. Correspondingly, groups that interact mainly by dispersive forces (CC, CH) were found to increase in intensity as takeoff angle increased, indicating they are segregated away from the DLC-protein interface. The magnitude of the segregation was seen to increase with increasing polar surface energy, this was attributed to an increased net attraction between the solid surface and polar groups at higher polar surface energy (γSp).

  12. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Protein adsorption at polymer-grafted surfaces: Comparison between a mixture of saliva proteins and some well-defined model proteins

    NARCIS (Netherlands)

    Kawasaki, K.; Kambara, M.; Matsumura, H.; Norde, W.

    2003-01-01

    Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The

  14. Controlled surface chemistry of diamond/β-SiC composite films for preferential protein adsorption.

    Science.gov (United States)

    Wang, Tao; Handschuh-Wang, Stephan; Yang, Yang; Zhuang, Hao; Schlemper, Christoph; Wesner, Daniel; Schönherr, Holger; Zhang, Wenjun; Jiang, Xin

    2014-02-04

    Diamond and SiC both process extraordinary biocompatible, electronic, and chemical properties. A combination of diamond and SiC may lead to highly stable materials, e.g., for implants or biosensors with excellent sensing properties. Here we report on the controllable surface chemistry of diamond/β-SiC composite films and its effect on protein adsorption. For systematic and high-throughput investigations, novel diamond/β-SiC composite films with gradient composition have been synthesized using the hot filament chemical vapor deposition (HFCVD) technique. As revealed by scanning electron microscopy (SEM), the diamond/β-SiC ratio of the composite films shows a continuous change from pure diamond to β-SiC over a length of ∼ 10 mm on the surface. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to unveil the surface termination of chemically oxidized and hydrogen treated surfaces. The surface chemistry of the composite films was found to depend on diamond/β-SiC ratio and the surface treatment. As observed by confocal fluorescence microscopy, albumin and fibrinogen were preferentially adsorbed from buffer: after surface oxidation, the proteins preferred to adsorb on diamond rather than on β-SiC, resulting in an increasing amount of proteins adsorbed to the gradient surfaces with increasing diamond/β-SiC ratio. By contrast, for hydrogen-treated surfaces, the proteins preferentially adsorbed on β-SiC, leading to a decreasing amount of albumin adsorbed on the gradient surfaces with increasing diamond/β-SiC ratio. The mechanism of preferential protein adsorption is discussed by considering the hydrogen bonding of the water self-association network to OH-terminated surfaces and the change of the polar surface energy component, which was determined according to the van Oss method. These results suggest that the diamond/β-SiC gradient film can be a promising material for biomedical applications which

  15. Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method.

    Science.gov (United States)

    Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

    2014-08-01

    Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

  16. CASTp 3.0: computed atlas of surface topography of proteins.

    Science.gov (United States)

    Tian, Wei; Chen, Chang; Lei, Xue; Zhao, Jieling; Liang, Jie

    2018-06-01

    Geometric and topological properties of protein structures, including surface pockets, interior cavities and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a web server that provides online services for locating, delineating and measuring these geometric and topological properties of protein structures. It has been widely used since its inception in 2003. In this article, we present the latest version of the web server, CASTp 3.0. CASTp 3.0 continues to provide reliable and comprehensive identifications and quantifications of protein topography. In addition, it now provides: (i) imprints of the negative volumes of pockets, cavities and channels, (ii) topographic features of biological assemblies in the Protein Data Bank, (iii) improved visualization of protein structures and pockets, and (iv) more intuitive structural and annotated information, including information of secondary structure, functional sites, variant sites and other annotations of protein residues. The CASTp 3.0 web server is freely accessible at http://sts.bioe.uic.edu/castp/.

  17. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  18. DBAC: A simple prediction method for protein binding hot spots based on burial levels and deeply buried atomic contacts

    Science.gov (United States)

    2011-01-01

    Background A protein binding hot spot is a cluster of residues in the interface that are energetically important for the binding of the protein with its interaction partner. Identifying protein binding hot spots can give useful information to protein engineering and drug design, and can also deepen our understanding of protein-protein interaction. These residues are usually buried inside the interface with very low solvent accessible surface area (SASA). Thus SASA is widely used as an outstanding feature in hot spot prediction by many computational methods. However, SASA is not capable of distinguishing slightly buried residues, of which most are non hot spots, and deeply buried ones that are usually inside a hot spot. Results We propose a new descriptor called “burial level” for characterizing residues, atoms and atomic contacts. Specifically, burial level captures the depth the residues are buried. We identify different kinds of deeply buried atomic contacts (DBAC) at different burial levels that are directly broken in alanine substitution. We use their numbers as input for SVM to classify between hot spot or non hot spot residues. We achieve F measure of 0.6237 under the leave-one-out cross-validation on a data set containing 258 mutations. This performance is better than other computational methods. Conclusions Our results show that hot spot residues tend to be deeply buried in the interface, not just having a low SASA value. This indicates that a high burial level is not only a necessary but also a more sufficient condition than a low SASA for a residue to be a hot spot residue. We find that those deeply buried atoms become increasingly more important when their burial levels rise up. This work also confirms the contribution of deeply buried interfacial atomic contacts to the energy of protein binding hot spot. PMID:21689480

  19. Nanoscale protein arrays of rich morphologies via self-assembly on chemically treated diblock copolymer surfaces

    International Nuclear Information System (INIS)

    Song Sheng; Milchak, Marissa; Zhou Hebing; Lee, Thomas; Hanscom, Mark; Hahm, Jong-in

    2013-01-01

    Well-controlled assembly of proteins on supramolecular templates of block copolymers can be extremely useful for high-throughput biodetection. We report the adsorption and assembly characteristics of a model antibody protein to various polystyrene-block-poly(4-vinylpyridine) templates whose distinctive nanoscale structures are obtained through time-regulated exposure to chloroform vapor. The strong adsorption preference of the protein to the polystyrene segment in the diblock copolymer templates leads to an easily predictable, controllable, rich set of nanoscale protein morphologies through self-assembly. We also demonstrate that the chemical identities of various subareas within individual nanostructures can be readily elucidated by investigating the corresponding protein adsorption behavior on each chemically distinct area of the template. In our approach, a rich set of intricate nanoscale morphologies of protein arrays that cannot be easily attained through other means can be generated straightforwardly via self-assembly of proteins on chemically treated diblock copolymer surfaces, without the use of clean-room-based fabrication tools. Our approach provides much-needed flexibility and versatility for the use of block copolymer-based protein arrays in biodetection. The ease of fabrication in producing well-defined and self-assembled templates can contribute to a high degree of versatility and simplicity in acquiring an intricate nanoscale geometry and spatial distribution of proteins in arrays. These advantages can be extremely beneficial both for fundamental research and biomedical detection, especially in the areas of solid-state-based, high-throughput protein sensing. (paper)

  20. Voltammetry and In Situ Scanning Tunnelling Microscopy of De Novo Designed Heme Protein Monolayers on Au(111)-Electrode Surfaces

    DEFF Research Database (Denmark)

    Albrecht, Tim; Li, Wu; Haehnel, Wolfgang

    2006-01-01

    to the tunnelling current, apparently due to slow electron transfer kinetics. As a consequence, STM images of heme-containing and heme-free MOP-C did not reveal any notable differences in apparent height or physical extension. The apparent height of heme-containing MOP-C did not show any dependence on the substrate...... potential being varied around the redox potential of the protein. The mere presence of an accessible molecular energy level is not sufficient to result in detectable tunnelling current modulation. (c) 2006 Elsevier B.V. All rights reserved.......In the present work, we report the electrochemical characterization and in situ scanning tunnelling microscopy (STM) studies of monolayers of an artificial de novo designed heme protein MOP-C, covalently immobilized on modified Au(111) surfaces. The protein forms closely packed monolayers, which...

  1. The effect of serum magnesium levels and serum endothelin-1 levels on bone mineral density in protein energy malnutrition.

    Science.gov (United States)

    Ozturk, C F; Karakelleoglu, C; Orbak, Z; Yildiz, L

    2012-06-01

    An inadequate and imbalanced intake of protein and energy results in protein-energy malnutrition (PEM). It is known that bone mineral density and serum magnesium levels are low in malnourished children. However, the roles of serum magnesium and endothelin-1 (ET-1) levels in the pathophysiology of bone mineralization are obscure. Thus, the relationships between serum magnesium and ET-1 levels and the changes in bone mineral density were investigated in this study. There was a total of 32 subjects, 25 of them had PEM and seven were controls. While mean serum ET-1 levels of the children with kwashiorkor and marasmus showed no statistically significant difference, mean serum ET-1 levels of both groups were significantly higher than that of the control group. Serum magnesium levels were lower than normal value in 9 (36%) of 25 malnourished children. Malnourished children included in this study were divided into two subgroups according to their serum magnesium levels. While mean serum ET-1 levels in the group with low magnesium levels were significantly higher than that of the group with normal magnesium levels (p malnutrition. Our study suggested that lower magnesium levels and higher ET-1 levels might be important factors in changes of bone mineral density in malnutrition. We recommend that the malnourished patients, especially with hypomagnesaemia, should be treated with magnesium early.

  2. Protein structural transition at negatively charged electrode surfaces. Effects of temperature and current density

    Czech Academy of Sciences Publication Activity Database

    Černocká, Hana; Ostatná, Veronika; Paleček, Emil

    2015-01-01

    Roč. 174, AUG 2015 (2015), s. 356-360 ISSN 0013-4686 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GA15-15479S; GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : Bovine serum albumin * sensing of surface-attached protein stability * protein structural transition at Hg Subject RIV: BO - Biophysics Impact factor: 4.803, year: 2015

  3. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    OpenAIRE

    Patricia A Martin-DeLeon

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the ...

  4. Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2.

    Directory of Open Access Journals (Sweden)

    Eva Herrero

    2016-02-01

    Full Text Available The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.

  5. Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer.

    Science.gov (United States)

    Wilts, I T; Hutten, B A; Meijers, J C M; Spek, C A; Büller, H R; Kamphuisen, P W

    2017-06-01

    Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. We analysed 477 patients (protein C tertiles: C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Protein levels and colony development of Africanized and European honey bees fed natural and artificial diets.

    Science.gov (United States)

    Morais, M M; Turcatto, A P; Pereira, R A; Francoy, T M; Guidugli-Lazzarini, K R; Gonçalves, L S; de Almeida, J M V; Ellis, J D; De Jong, D

    2013-12-19

    Pollen substitute diets are a valuable resource for maintaining strong and health honey bee colonies. Specific diets may be useful in one region or country and inadequate or economically unviable in others. We compared two artificial protein diets that had been formulated from locally-available ingredients in Brazil with bee bread and a non-protein sucrose diet. Groups of 100 newly-emerged, adult workers of Africanized honey bees in Brazil and European honey bees in the USA were confined in small cages and fed on one of four diets for seven days. The artificial diets included a high protein diet made of soy milk powder and albumin, and a lower protein level diet consisting of soy milk powder, brewer's yeast and rice bran. The initial protein levels in newly emerged bees were approximately 18-21 µg/µL hemolymph. After feeding on the diets for seven days, the protein levels in the hemolymph were similar among the protein diet groups (~37-49 µg/µL after seven days), although Africanized bees acquired higher protein levels, increasing 145 and 100% on diets D1 and D2, respectively, versus 83 and 60% in the European bees. All the protein diets resulted in significantly higher levels of protein than sucrose solution alone. In the field, the two pollen substitute diets were tested during periods of low pollen availability in the field in two regions of Brazil. Food consumption, population development, colony weight, and honey production were evaluated to determine the impact of the diets on colony strength parameters. The colonies fed artificial diets had a significant improvement in all parameters, while control colonies dwindled during the dearth period. We conclude that these two artificial protein diets have good potential as pollen substitutes during dearth periods and that Africanized bees more efficiently utilize artificial protein diets than do European honey bees.

  7. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

  8. Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

    OpenAIRE

    Suits, Michael D.; Boraston, Alisdair B.

    2013-01-01

    PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-he...

  9. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

    Science.gov (United States)

    Shirvan, Ali Nazari; Aitken, Robert

    2016-01-01

    Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

  10. Analysis of the free-energy surface of proteins from reversible folding simulations.

    Directory of Open Access Journals (Sweden)

    Lucy R Allen

    2009-07-01

    Full Text Available Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical lambda-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins.

  11. human serum protein and c-reactive protein levels among hiv ...

    African Journals Online (AJOL)

    2016-09-30

    Sep 30, 2016 ... inflammation used to monitor HIV infection (Pepys and Hirschfield, 2003; Baker et al., 2010; Funderburg et al., 2010;. Neuhaus et ... from microbial infections, the CRP concentration can rise up to 300mg/L in 12-24 hours (Le Carrer et al., 1995; Vaishnavi,. 1996 ..... (pentaxins) and serum amyloid A protein.

  12. Animal Protein intake and the Effect of age on the level of Protein ...

    African Journals Online (AJOL)

    Some of the items such as eggs, milk and poultry meat were not consumed in reasonable quantities due to poor economic situation of the family. The results suggested that except for the fathers, other groups such as mothers and children were consuming animal protein below the quantity required by them. JARD Vol.

  13. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

    Science.gov (United States)

    Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

    2005-01-01

    Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  14. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  15. Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

    Science.gov (United States)

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

  16. Protein-coated pH-responsive gold nanoparticles: Microwave-assisted synthesis and surface charge-dependent anticancer activity

    Directory of Open Access Journals (Sweden)

    Dickson Joseph

    2014-09-01

    Full Text Available The biocompatibility and ease of functionalization of gold nanoparticles underlie significant potential in biotechnology and biomedicine. Eight different proteins were examined in the preparation of gold nanoparticles (AuNPs in aqueous medium under microwave irradiation. Six of the proteins resulted in the formation of AuNPs. The intrinsic pH of the proteins played an important role in AuNPs with strong surface plasmon bands. The hydrodynamic size of the nanoparticles was larger than the values observed by TEM and ImageJ. The formation of a protein layer on the AuNPs accounts for this difference. The AuNPs exhibited sensitivity towards varying pH conditions, which was confirmed by determining the difference in the isoelectric points studied by using pH-dependent zeta potential titration. Cytotoxicity studies revealed anticancerous effects of the AuNPs at a certain micromolar concentration by constraining the growth of cancer cells with different efficacies due to the use of different proteins as capping agents. The positively charged AuNPs are internalized by the cells to a greater level than the negatively charged AuNPs. These AuNPs synthesized with protein coating holds promise as anticancer agents and would help in providing a new paradigm in area of nanoparticles.

  17. Identification of Cell Surface Proteins as Potential Immunotherapy Targets in 12 Pediatric Cancers

    Energy Technology Data Exchange (ETDEWEB)

    Orentas, Rimas J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Yang, James J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Wen, Xinyu; Wei, Jun S. [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Mackall, Crystal L. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Khan, Javed, E-mail: rimas.orentas@nih.gov [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States)

    2012-12-17

    Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

  18. Optimising the Use of TRIzol-extracted Proteins in Surface Enhanced Laser Desorption/ Ionization (SELDI Analysis

    Directory of Open Access Journals (Sweden)

    Perlaky Laszlo

    2006-03-01

    Full Text Available Abstract Background Research with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization. Results To facilitate the use of TRIzol-extracted proteins, we first compared the ability of four different common solubilizing reagents to solubilize the TRIzol-extracted proteins from an osteosarcoma cell line, U2-OS. Then we analyzed the solubilized proteins by Surface Enhanced Laser Desorption/ Ionization technique (SELDI. The results showed that solubilization of TRIzol-extracted proteins with 9.5 M Urea and 2% CHAPS ([3-[(3-cholamidopropyl-dimethylammonio]propanesulfonate] (UREA-CHAPS was significantly better than the standard 1% SDS in terms of solubilization efficiency and the number of detectable ion peaks. Using three different types of SELDI arrays (CM10, H50, and IMAC-Cu, we demonstrated that peak detection with proteins solubilized by UREA-CHAPS was reproducible (r > 0.9. Further SELDI analysis indicated that the number of ion peaks detected in TRIzol-extracted proteins was comparable to a direct extraction method, suggesting many proteins still remain in the TRIzol protein fraction. Conclusion Our results suggest that UREA-CHAPS performed very well in solubilizing TRIzol-extracted proteins for SELDI applications. Protein fractions left over after TRIzol RNA extraction could be a valuable but neglected source for proteomic or biochemical analysis when additional samples are not available.

  19. Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates

    Science.gov (United States)

    Martin, Nicholas J.; Griffiths, Rian L.; Edwards, Rebecca L.; Cooper, Helen J.

    2015-08-01

    Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The `contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

  20. Origin of cell surface proteins released from Micrococcus radiodurans by ionizing radiation

    International Nuclear Information System (INIS)

    Mitchel, R.E.J.

    1975-01-01

    The exposure of Micrococcus radiodurans to sublethal doses of ionizing radiation causes the release of certain proteins into the surrounding medium. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these proteins range from approximately 20,000 to 125,000 daltons. At least some of the proteins, including an exonuclease, have a surface location and appear to originate from the lipid-rich midwall layer. The exonuclease has two functionally distinct locations, one with its active site available to external substrate and a second with the active site masked from the exterior. Ionizing radiation releases both the masked and unmasked activity into the surrounding medium

  1. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    OpenAIRE

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2015-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP....

  2. Predicting Ligand Binding Sites on Protein Surfaces by 3-Dimensional Probability Density Distributions of Interacting Atoms

    Science.gov (United States)

    Jian, Jhih-Wei; Elumalai, Pavadai; Pitti, Thejkiran; Wu, Chih Yuan; Tsai, Keng-Chang; Chang, Jeng-Yih; Peng, Hung-Pin; Yang, An-Suei

    2016-01-01

    Predicting ligand binding sites (LBSs) on protein structures, which are obtained either from experimental or computational methods, is a useful first step in functional annotation or structure-based drug design for the protein structures. In this work, the structure-based machine learning algorithm ISMBLab-LIG was developed to predict LBSs on protein surfaces with input attributes derived from the three-dimensional probability density maps of interacting atoms, which were reconstructed on the query protein surfaces and were relatively insensitive to local conformational variations of the tentative ligand binding sites. The prediction accuracy of the ISMBLab-LIG predictors is comparable to that of the best LBS predictors benchmarked on several well-established testing datasets. More importantly, the ISMBLab-LIG algorithm has substantial tolerance to the prediction uncertainties of computationally derived protein structure models. As such, the method is particularly useful for predicting LBSs not only on experimental protein structures without known LBS templates in the database but also on computationally predicted model protein structures with structural uncertainties in the tentative ligand binding sites. PMID:27513851

  3. A Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis**

    OpenAIRE

    Grover, Anmol; Schmidt, Brigitte F.; Salter, Russell D.; Watkins, Simon C.; Waggoner, Alan S.; Bruchez, Marcel P.

    2012-01-01

    We have combined our fluorogen activating peptide[1] with a new tandem dye molecule to develop a biosensor that labels a cell-surface protein and displays an easily detectable pH dependent emission color change by efficient intramolecular Förster resonant energy transfer. This probe has demonstrated pH variations in β2-adrenergic receptor trafficking and revealed a process of surface to endosome inter-cellular transfer in dendritic cells with potential significance in antigen transfer.

  4. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

    Science.gov (United States)

    Lu, Ming-Chi; Yu, Chia-Li; Yu, Hui-Chun; Huang, Hsien-Bin; Koo, Malcolm; Lai, Ning-Sheng

    2016-01-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

    Directory of Open Access Journals (Sweden)

    Chang T-H

    2006-01-01

    Full Text Available Abstract Background Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD that affects more than 250 million people worldwide. Immunoglobulin A (IgA has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb immunoreactive to trichomonads by whole cell (WC-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis. Results An IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44 by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44 of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs. Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax. Conclusion This is the first report of a T. vaginalis gene (tv44 encoding a surface protein (TV44 reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs.

  6. Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

    International Nuclear Information System (INIS)

    Dånmark, S; Mustafa, K; Finne-Wistrand, A; Albertsson, A-C; Patarroyo, M

    2012-01-01

    In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin. (paper)

  7. Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites.

    Science.gov (United States)

    Zhang, Deqing; Howe, Daniel K

    2008-04-15

    An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.

  8. Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

    International Nuclear Information System (INIS)

    Nestler, J.E.; McLeod, J.F.; Kowalski, M.A.; Strauss, J.F. III; Haddad, J.G. Jr.

    1987-01-01

    Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [ 35 S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated

  9. Multi-level learning: improving the prediction of protein, domain and residue interactions by allowing information flow between levels

    Directory of Open Access Journals (Sweden)

    McDermott Drew

    2009-08-01

    Full Text Available Abstract Background Proteins interact through specific binding interfaces that contain many residues in domains. Protein interactions thus occur on three different levels of a concept hierarchy: whole-proteins, domains, and residues. Each level offers a distinct and complementary set of features for computationally predicting interactions, including functional genomic features of whole proteins, evolutionary features of domain families and physical-chemical features of individual residues. The predictions at each level could benefit from using the features at all three levels. However, it is not trivial as the features are provided at different granularity. Results To link up the predictions at the three levels, we propose a multi-level machine-learning framework that allows for explicit information flow between the levels. We demonstrate, using representative yeast interaction networks, that our algorithm is able to utilize complementary feature sets to make more accurate predictions at the three levels than when the three problems are approached independently. To facilitate application of our multi-level learning framework, we discuss three key aspects of multi-level learning and the corresponding design choices that we have made in the implementation of a concrete learning algorithm. 1 Architecture of information flow: we show the greater flexibility of bidirectional flow over independent levels and unidirectional flow; 2 Coupling mechanism of the different levels: We show how this can be accomplished via augmenting the training sets at each level, and discuss the prevention of error propagation between different levels by means of soft coupling; 3 Sparseness of data: We show that the multi-level framework compounds data sparsity issues, and discuss how this can be dealt with by building local models in information-rich parts of the data. Our proof-of-concept learning algorithm demonstrates the advantage of combining levels, and opens up

  10. Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

    Science.gov (United States)

    Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

    2015-01-01

    Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

    Science.gov (United States)

    Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

    2013-03-01

    Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

  12. Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

    Science.gov (United States)

    Zhang, Xi

    2015-01-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

  13. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  14. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32

    Directory of Open Access Journals (Sweden)

    Sugiyama Hideaki

    2011-05-01

    Full Text Available Abstract Background Elevated numbers of regulatory T cells (Tregs have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32, also known as Glycoprotein A Repetitions Predominant (GARP, has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  15. Genetic regulation ofmethylation and IL1RL1-a protein levels in asthma

    NARCIS (Netherlands)

    Dijk, F Nicole; Xu, Chengjian; Melén, Erik; Carsin, Anne-Elie; Kumar, Asish; Nolte, Ilja M; Gruzieva, Olena; Pershagen, Goran; Grotenboer, Neomi S; Savenije, Olga E M; Antó, Josep Maria; Lavi, Iris; Dobaño, Carlota; Bousquet, Jean; van der Vlies, Pieter; van der Valk, Ralf J P; de Jongste, Johan C; Nawijn, Martijn C; Guerra, Stefano; Postma, Dirkje S; Koppelman, Gerard H

    2018-01-01

    Interleukin-1 receptor-like 1 (IL1RL1) is an important asthma gene. (Epi)genetic regulation ofIL1RL1protein expression has not been established. We assessed the association betweenIL1RL1single nucleotide polymorphisms (SNPs),IL1RL1methylation and serum IL1RL1-a protein levels, and aimed to identify

  16. Effect of different protein levels on the growth performance of African ...

    African Journals Online (AJOL)

    There were no significant differences (P.0.05) among treatments in initial body weight, protein efficiency ratio, average shell weight, average visceral weight, average edible weight and feed cost per kg weight gain. The results obtained in this study show that dietary protein level of about 22% is adequate for the growth of ...

  17. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

    Science.gov (United States)

    Martin-DeLeon, Patricia A

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  18. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    Directory of Open Access Journals (Sweden)

    Patricia A Martin-DeLeon

    2015-01-01

    Full Text Available A variety of glycosylphosphatidylinositol (GPI-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4. Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4′s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  19. Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

    Science.gov (United States)

    Taupin, Philippe

    2011-09-01

    The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

  20. Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma

    Czech Academy of Sciences Publication Activity Database

    Piliarik, Marek; Bocková, Markéta; Homola, Jiří

    2010-01-01

    Roč. 26, č. 4 (2010), s. 1656-1661 ISSN 0956-5663 R&D Projects: GA AV ČR KAN200670701 Institutional research plan: CEZ:AV0Z20670512 Keywords : Surface plasmon resonance * Protein array * Cancer marker Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 5.361, year: 2010

  1. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    International Nuclear Information System (INIS)

    Lu Yan; Yan Changling; Gao Shuyan

    2009-01-01

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  2. Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry

    NARCIS (Netherlands)

    Bouchon, B.; Klein, Michele; Bischoff, Rainer; Van Dorsselaer, A.; Roitsch, C.

    1997-01-01

    The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da

  3. Functionalization of SU-8 photoresist surfaces with IgG proteins

    International Nuclear Information System (INIS)

    Blagoi, Gabriela; Keller, Stephan; Johansson, Alicia; Boisen, Anja; Dufva, Martin

    2008-01-01

    The negative epoxy-based photoresist SU-8 has a variety of applications within microelectromechanical systems (MEMS) and lab-on-a-chip systems. Here, several methods to functionalize SU-8 surfaces with IgG proteins were investigated. Fluorescent labeled proteins and fluorescent sandwich immunoassays were employed to characterize the binding efficiency of model proteins to bare SU-8 surface, SU-8 treated with cerium ammonium nitrate (CAN) etchant and CAN treated surfaces modified by aminosilanization. The highest binding capacity of antibodies was observed on bare SU-8. This explains why bare SU-8 in a functional fluorescent sandwich immunoassay detecting C-reactive protein (CRP) gave twice as high signal as compared with the other two surfaces. Immunoassays performed on bare SU-8 and CAN treated SU-8 resulted in detection limits of CRP of 30 and 80 ng/ml respectively which is sufficient for detecting CRP in clinical samples, where concentrations of 3-10 μg/ml are normal for healthy individuals. In conclusion, bare SU-8 and etched SU-8 can be modified with antibodies by a simple adsorption procedure which simplifies building lab-on-a-chip systems in SU-8. Additionally, we report the fabrication process and use of microwells created in a SU-8 layer with the same dimensions as a standard microscope glass slide that could fit into fluorescent scanners. The SU-8 microwells minimize the reagent consumption and are straightforward to handle compared to SU-8 coated microscope slides

  4. Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension.

    Science.gov (United States)

    Amrhein, Sven; Bauer, Katharina Christin; Galm, Lara; Hubbuch, Jürgen

    2015-12-01

    The surface hydrophobicity of a protein is an important factor for its interactions in solution and thus the outcome of its production process. Yet most of the methods are not able to evaluate the influence of these hydrophobic interactions under natural conditions. In the present work we have established a high resolution stalagmometric method for surface tension determination on a liquid handling station, which can cope with accuracy as well as high throughput requirements. Surface tensions could be derived with a low sample consumption (800 μL) and a high reproducibility (content. The protein influence on the solutions' surface tension was correlated to the hydrophobicity of lysozyme, human lysozyme, BSA, and α-lactalbumin. Differences in proteins' hydrophobic character depending on pH and species could be resolved. Within this work we have developed a pH dependent hydrophobicity ranking, which was found to be in good agreement with literature. For the studied pH range of 3-9 lysozyme from chicken egg white was identified to be the most hydrophilic. α-lactalbumin at pH 3 exhibited the most pronounced hydrophobic character. The stalagmometric method occurred to outclass the widely used spectrophotometric method with bromophenol blue sodium salt as it gave reasonable results without restrictions on pH and protein species. © 2015 Wiley Periodicals, Inc.

  5. Enterococcal surface protein transiently aggravates Enterococcus faecium-induced urinary tract infection in mice

    NARCIS (Netherlands)

    Leendertse, Masja; Heikens, Esther; Wijnands, Lucas M.; van Luit-Asbroek, Miranda; Teske, Gwendoline J. D.; Roelofs, Joris J. T. H.; Bonten, Marc J. M.; van der Poll, Tom; Willems, Rob J. L.

    2009-01-01

    The role that the enterococcal surface protein Esp plays in the capacity of Enterococcus faecium to adhere to uroepithelial cells and the role that it plays in urinary tract infection and peritonitis was investigated in vitro and in vivo, respectively, using Esp-expressing E. faecium (E1162) and its

  6. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China); Yan Changling; Gao Shuyan [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China)

    2009-04-01

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  7. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Czech Academy of Sciences Publication Activity Database

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    2016-01-01

    Roč. 132, č. 1 (2016), s. 13-20 ISSN 1874-3919 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.914, year: 2016

  8. Glioblastoma Inhibition by Cell Surface Immunoglobulin Protein EWI-2, In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana V. Kolesnikova

    2009-01-01

    Full Text Available EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP, which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.

  9. 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.

  10. Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

    Science.gov (United States)

    Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

    2001-08-01

    The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

  11. Sea level and turbidity controls on mangrove soil surface elevation change

    Science.gov (United States)

    Lovelock, Catherine E.; Fernanda Adame, Maria; Bennion, Vicki; Hayes, Matthew; Reef, Ruth; Santini, Nadia; Cahoon, Donald R.

    2015-01-01

    Increases in sea level are a threat to seaward fringing mangrove forests if levels of inundation exceed the physiological tolerance of the trees; however, tidal wetlands can keep pace with sea level rise if soil surface elevations can increase at the same pace as sea level rise. Sediment accretion on the soil surface and belowground production of roots are proposed to increase with increasing sea level, enabling intertidal habitats to maintain their position relative to mean sea level, but there are few tests of these predictions in mangrove forests. Here we used variation in sea level and the availability of sediments caused by seasonal and inter-annual variation in the intensity of La Nina-El Nino to assess the effects of increasing sea level on surface elevation gains and contributing processes (accretion on the surface, subsidence and root growth) in mangrove forests. We found that soil surface elevation increased with mean sea level (which varied over 250 mm during the study) and with turbidity at sites where fine sediment in the water column is abundant. In contrast, where sediments were sandy, rates of surface elevation gain were high, but not significantly related to variation in turbidity, and were likely to be influenced by other factors that deliver sand to the mangrove forest. Root growth was not linked to soil surface elevation gains, although it was associated with reduced shallow subsidence, and therefore may contribute to the capacity of mangroves to keep pace with sea level rise. Our results indicate both surface (sedimentation) and subsurface (root growth) processes can influence mangrove capacity to keep pace with sea level rise within the same geographic location, and that current models of tidal marsh responses to sea level rise capture the major feature of the response of mangroves where fine, but not coarse, sediments are abundant.

  12. Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

    DEFF Research Database (Denmark)

    Sultan, Abida; Andersen, Birgit; Svensson, Birte

    2016-01-01

    Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant...... xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC–MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface......-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation...

  13. Fluorescent proteins as efficient tools for evaluating the surface PEGylation of silica nanoparticles

    Science.gov (United States)

    Zhang, Wei; Ma, Minyan; Zhang, Xiao-ai; Zhang, Ze-yu; Saleh, Sayed M.; Wang, Xu-dong

    2017-06-01

    Surface PEGylation is essential for preventing non-specific binding of biomolecules when silica nanoparticles are utilized for in vivo applications. Methods for installing poly(ethylene glycol) on a silica surface have been widely explored but varies from study to study. Because there is a lack of a satisfactory method for evaluating the properties of silica surface after PEGylation, the prepared nanoparticles are not fully characterized before use. In some cases, even non-PEGylated silica nanoparticles were produced, which is unfortunately not recognized by the end-user. In this work, a fluorescent protein was employed, which acts as a sensitive material for evaluating the surface protein adsorption properties of silica nanoparticles. Eleven different methods were systematically investigated for their reaction efficiency towards surface PEGylation. Results showed that both reaction conditions (including pH, catalyst) and surface functional groups of parent silica nanoparticles play critical roles in producing fully PEGylated silica nanoparticles. Great care needs to be taken in choosing the proper coupling chemistry for surface PEGylation. The data and method shown here will guarantee high-quality PEGylated silica nanoparticles to be produced and guide their applications in biology, chemistry, industry and medicine.

  14. Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

    International Nuclear Information System (INIS)

    Tilley, M.; Upton, S.J.

    1990-01-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and 125 I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic

  15. Assessment of surface contamination level in an operating uranium ore processing facility of Jaduguda, India

    International Nuclear Information System (INIS)

    Meena, J.S.; Patnaik, R.L.; Jha, V.N.; Sahoo, S.K.; Ravi, P.M.; Tripathi, R.M.

    2014-01-01

    Radiological concern of the occupational workers and the area is given priority over other safety issue in confirmation with the stipulated guideline of national regulatory agency (AERB/FEFCF/SG-2, 2007). The key concern from the radiological hazard evaluation point of view is air activity, external gamma level and surface contamination. Present investigations was carried out to ascertain the surface contamination level of uranium ore processing facility at Jaduguda, Jharkhand. For a low grade uranium ore processing industry surface contamination is a major concern in product precipitation and recovery section. In view of this, the ore processing plant can broadly be classified into three areas i.e. ion exchange area, precipitation and product recovery section and other areas. The monitoring results incorporate the level of surface contamination of the plant during the last five years. The geometric mean activity of surface contamination level was 31.1, 34.5 and 9.8 Bq dm -2 in ion exchange, product precipitation and recovery and other areas with GSD of 2, 2.5 and 1.9. In most of the cases the surface contamination level was well within the recommended limit of 100 Bq dm -2 for M class uranium compound. Occasional cases of surface contamination levels exceeding the recommended limit were addressed and areas were decontaminated. Based on the study, modification in the design feature of the surface of the finished product section was also suggested so that the decontamination procedure can be more effectively implemented

  16. THE SURFACE-MEDIATED UNFOLDING KINETICS OF GLOBULAR PROTEINS IS DEPENDENT ON MOLECULAR WEIGHT AND TEMPERATURE

    Energy Technology Data Exchange (ETDEWEB)

    Patananan, A.N.; Goheen, S.C.

    2008-01-01

    The adsorption and unfolding pathways of proteins on rigid surfaces are essential in numerous complex processes associated with biomedical engineering, nanotechnology, and chromatography. It is now well accepted that the kinetics of unfolding are characterized by chemical and physical interactions dependent on protein deformability and structure, as well as environmental pH, temperature, and surface chemistry. Although this fundamental process has broad implications in medicine and industry, little is known about the mechanism because of the atomic lengths and rapid time scales involved. Therefore, the unfolding kinetics of myoglobin, β-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, β-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and β-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface

  17. Nitrogen balance study in young Nigerian adult males using four levels of protein intake.

    Science.gov (United States)

    Atinmo, T; Mbofung, C M; Egun, G; Osotimehin, B

    1988-11-01

    1. The present study was carried out to estimate precisely, via the nitrogen balance technique, the protein requirement of Nigerians (earlier estimated via the obligatory N method) using graded levels of protein intake. 2. Fifteen medical students of the University of Ibadan who volunteered to participate in the study were given graded levels of protein (0.3, 0.45, 0.6 and 0.75 g/kg body-weight per d) derived from foods similar to those usually consumed by the subjects. 3. Each subject was given each of the dietary protein levels for a period of 10 d. Subjects were divided into two groups and the feeding pattern followed a criss-cross design with one group starting with the highest level of protein intake (0.3 g). Mean energy intake during each of the eleven experimental periods was maintained at 0.2 MJ/kg per d. After an initial 5 d adaptation period in each experimental period, 24 h urine and faecal samples were collected in marked containers for five consecutive days for N determination. 4. Mean N balance during consumption of the four protein levels (0.30, 0.45, 0.6 and 0.75 g/kg) were -11.02 (SD 8.07), -9.90 (SD 6.64), +9.70 (SD 4.15) and +5.13 (SD 4.62) respectively. Using regression analysis, the mean daily N requirement was estimated at 110.25 mg N/kg body-weight (0.69 g protein/kg body-weight). Estimates of allowances for individual variations to cover 97.5% of the population adjusted this value to 0.75 g protein/kg body-weight. Net protein utilization for the diet at maintenance level was estimated at 57.5.

  18. Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

    Science.gov (United States)

    Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

    2005-01-01

    Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

  19. Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

    Science.gov (United States)

    Ba, Xiaolan

    biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

  20. Functionalization of SU-8 Photoresist Surfaces with IgG Proteins

    DEFF Research Database (Denmark)

    Blagoi, Gabriela; Keller, Stephan Urs; Johansson, Alicia

    2008-01-01

    immunoassays were employed to characterize the binding efficiency of model proteins to bare SU-8 surface, SU-8 treated with cerium ammonium nitrate (CAN) etchant and CAN treated surfaces modified by aminosilanization. The highest binding capacity of antibodies was observed on bare SU-8. This explains why bare...... SU-8 in a functional fluorescent sandwich immunoassay detecting C-reactive protein (CRP) gave twice as high signal as compared with the other two surfaces. Immunoassays performed on bare SU-8 and CAN treated SU-8 resulted in detection limits of CRP of 30 and 80 ng/ml respectively which is sufficient...... for detecting CRP in clinical samples, where concentrations of 3–10 μg/ml are normal for healthy individuals. In conclusion, bare SU-8 and etched SU-8 can be modified with antibodies by a simple adsorption procedure which simplifies building lab-on-a-chip systems in SU-8. Additionally, we report the fabrication...

  1. Protein intake and stress levels in nurses and housewives of Pakistan

    Science.gov (United States)

    Wattoo, Feroza Hamid; Memon, Muhammad Saleh; Memon, Allah Nawaz; Wattoo, Muhammad Hamid Sarwar; Asad, Muhammad Javaid; Siddique, Farzana

    2011-01-01

    Stress has many biological effects on human daily life. In the present study, dietary protein intake was correlated with the investigated stress levels of nurses and housewives of the targeted urban population. Age group ranged from 30 to 45 years and both the groups belonged to middle socioeconomic status. After calculations of environmental, psychological and physiological stresses, it was observed that the levels of stress in housewives were significantly higher than those of nurses. Recommended dietary allowances, RDA and actual protein intakes, API were also compared in both the groups. The found protein intake was less in housewives as compared to that of nurses. PMID:23961140

  2. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  3. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

    Science.gov (United States)

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-07-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d).

  4. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  5. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

    OpenAIRE

    Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

    2012-01-01

    We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedde...

  6. Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.

    Science.gov (United States)

    Ohvo-Rekilä, Henna; Mattjus, Peter

    2011-01-01

    The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Heightened systemic levels of neutrophil and eosinophil granular proteins in pulmonary tuberculosis and reversal following treatment.

    Science.gov (United States)

    Moideen, Kadar; Kumar, Nathella Pavan; Nair, Dina; Banurekha, Vaithilingam V; Bethunaickan, Ramalingam; Babu, Subash

    2018-04-09

    Granulocytes are activated during tuberculosis (TB) infection and act as immune effector cells and granulocyte responses are implicated in TB pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in latent TB (LTB) individuals. Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, and proteinase-3; major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these parameters in PTB individuals following anti-tuberculosis (ATT) treatment. Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, human proteinase 3 as well as MBP and EDN in comparison to LTB individuals. Our data also reveal that ATT resulted in reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase-3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and therefore, the pathogenesis of pulmonary TB. Copyright © 2018 American Society for Microbiology.

  8. Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Gordaliza, Estefanía, E-mail: emorenog@ucm.es [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040, Madrid (Spain); Stigter, Edwin C.A. [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Molecular Cancer Research, Universitair Medisch Centrum Utrecht, Wilhelmina Kinder Ziekenhuis, Lundlaan 6, 3584, EA Utrecht (Netherlands); Lindenburg, Petrus W.; Hankemeier, Thomas [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands)

    2016-06-07

    A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10{sup −9} m{sup 2} V{sup −1} s{sup −1}) when compared with unmodified fused silica (5.9 ± 0.1 10{sup −8} m{sup 2} V{sup −1} s{sup −1}). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use. - Highlights: • New coating using recrystallized surface-layer proteins on

  9. Changes in human parotid salivary protein and sialic acid levels during pregnancy.

    Science.gov (United States)

    D'Alessandro, S; Curbelo, H M; Tumilasci, O R; Tessler, J A; Houssay, A B

    1989-01-01

    Saliva was collected with a Carlson-Crittenden device, under citric acid stimulation, in 107 pregnant women, 9 puerperal and 7 non-pregnant controls. No significant changes were found in salivary flow rate, pH and amylase levels. The total protein levels were decreased during pregnancy and the puerperium. The sialic acid levels decreased gradually but markedly during pregnancy, returning to normal levels in the puerperium. These changes in parotid saliva may be related to the hormonal changes of pregnancy.

  10. Effects of atomic-level nano-structured hydroxyapatite on adsorption of bone morphogenetic protein-7 and its derived peptide by computer simulation.

    Science.gov (United States)

    Wang, Qun; Wang, Menghao; Lu, Xiong; Wang, Kefeng; Fang, Liming; Ren, Fuzeng; Lu, Guoming

    2017-11-09

    Hydroxyapatite (HA) is the principal inorganic component of bones and teeth and has been widely used as a bone repair material because of its good biocompatibility and bioactivity. Understanding the interactions between proteins and HA is crucial for designing biomaterials for bone regeneration. In this study, we evaluated the effects of atomic-level nano-structured HA (110) surfaces on the adsorption of bone morphogenetic protein-7 (BMP-7) and its derived peptide (KQLNALSVLYFDD) using molecular dynamics and density functional theory methods. The results indicated that the atomic-level morphology of HA significantly affected the interaction strength between proteins and HA substrates. The interactions of BMP-7 and its derived peptide with nano-concave and nano-pillar HA surfaces were stronger than those with flat or nano-groove HA surfaces. The results also revealed that if the groove size of nano-structured HA surfaces matched that of residues in the protein or peptide, these residues were likely to spread into the grooves of the nano-groove, nano-concave, and nano-pillar HA, further strengthening the interactions. These results are helpful in better understanding the adsorption behaviors of proteins onto nano-structured HA surfaces, and provide theoretical guidance for designing novel bioceramic materials for bone regeneration and tissue engineering.

  11. Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets

    Directory of Open Access Journals (Sweden)

    Ashford Paul

    2012-03-01

    Full Text Available Abstract Background Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. Results We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i analysis of a kinase superfamily highlights the

  12. Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets.

    Science.gov (United States)

    Ashford, Paul; Moss, David S; Alex, Alexander; Yeap, Siew K; Povia, Alice; Nobeli, Irene; Williams, Mark A

    2012-03-14

    Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i) analysis of a kinase superfamily highlights the conserved occurrence of surface pockets at the active

  13. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    Directory of Open Access Journals (Sweden)

    Mamgain Hitesh

    2009-10-01

    Full Text Available Abstract Background Imaging tools such as scanning electron microscope (SEM and atomic force microscope (AFM can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK 293, human breast cancer (MCF-7 and mouse melanoma (B16F1 cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM and scanning electron microscopy (SEM. The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor

  14. Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco-adhesion in the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    Muriel Mercier-Bonin

    2017-11-01

    Full Text Available Food and probiotic bacteria, in particular lactic acid bacteria, are ingested in large amounts by humans and are part of the transient microbiota which is increasingly considered to be able to impact the resident microbiota and thus possibly the host health. The lactic acid bacterium Lactococcus lactis is extensively used in starter cultures to produce dairy fermented food. Also because of a generally recognized as safe status, L. lactis has been considered as a possible vehicle to deliver in vivo therapeutic molecules with anti-inflammatory properties in the gastrointestinal tract. One of the key factors that may favor health effects of beneficial bacteria to the host is their capacity to colonize transiently the gut, notably through close interactions with mucus, which covers and protects the intestinal epithelium. Several L. lactis strains have been shown to exhibit mucus-binding properties and bacterial surface proteins have been identified as key determinants of such capacity. In this review, we describe the different types of surface proteins found in L. lactis, with a special focus on mucus-binding proteins and pili. We also review the different approaches used to investigate the adhesion of L. lactis to mucus, and particularly to mucins, one of its major components, and we present how these approaches allowed revealing the role of surface proteins in muco-adhesion.

  15. Enhanced protein loading on a planar Si(111)-H surface with second generation NTA

    Science.gov (United States)

    Liu, Xiang; Han, Huan-Mei; Liu, Hong-Bo; Xiao, Shou-jun

    2010-08-01

    A Si(111)-H surface was modified via a direct reaction between Si-H and 1-undecylenic acid (UA) under microwave irradiation to form molecular monolayers with terminal carboxyl groups. After esterifying carboxylic acid being esterified with N-hydroxysuccinimide (NHS), aminobutyl nitrilotriacetic acid (ANTA) was bound to the silicon surface through amidation (pH = 8.0) between its primary amino group and NHS-ester, producing nitrilotriacetic acid (NTA) anions. Then hexa-histidine tagged thioredoxin-urodilatin (his-tagged protein) and FITC-labeled hexa-histidine tagged thioredoxin-urodilatin (FITC-his-tagged protein) can be anchored after NTA was coordinated with Ni 2+. Furthermore, the NTA-terminated chip was acidified with 0.1 M HCl and subsequently esterified with NHS and then amidated with ANTA again to produce a second generation NTA. Thus the surface density of nitrilotriacetic acid anions was improved and resultantly that of anchored proteins was also enhanced through the iterative reactions. Both multiple transmission-reflection infrared spectroscopy (MTR-IR) and fluorescence scanning measurements demonstrated a proximate 1.63 times of anchored proteins on the second generation NTA/Ni 2+ as that on the first generation NTA/Ni 2+ monolayer.

  16. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  17. Fasting Lipoprotein Lipase Protein Levels Can Predict a Postmeal Increment of Triglyceride Levels in Fasting Normohypertriglyceridemic Subjects.

    Science.gov (United States)

    Tsuzaki, Kokoro; Kotani, Kazuhiko; Yamada, Kazunori; Sakane, Naoki

    2016-09-01

    Although a postprandial increment in triglyceride (TG) levels is considered to be a risk factor for atherogenesis, tests (e.g., fat load) to assess postprandial changes in TG levels cannot be easily applied to clinical practice. Therefore, fasting markers that predict postprandial TG states are needed to be developed. One current candidate is lipoprotein lipase (LPL) protein, a molecule that hydrides TGs. This study investigated whether fasting LPL levels could predict postprandial TG levels. A total of 17 subjects (11 men, 6 women, mean age 52 ± 11 years) with normotriglyceridemia during fasting underwent the meal test. Several fasting parameters, including LPL, were measured for the area under the curve of postprandial TGs (AUC-TG). The subjects' mean fasting TG level was 1.30 mmol/l, and their mean LPL level was 41.6 ng/ml. The subjects' TG levels increased after loading (they peaked after two postprandial hours). Stepwise multiple regression analysis demonstrated that fasting TG levels were a predictor of the AUC-TG. In addition, fasting LPL mass levels were found to be a predictor of the AUC-TG (β = 0.65, P fasting TG levels. Fasting LPL levels may be useful to predict postprandial TG increment in this population. © 2015 Wiley Periodicals, Inc.

  18. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    Energy Technology Data Exchange (ETDEWEB)

    Burcza, Anna, E-mail: anna.burcza@mri.bund.de; Gräf, Volker; Walz, Elke; Greiner, Ralf [Max Rubner-Institute, Department of Food Technology and Bioprocess Engineering (Germany)

    2015-11-15

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated.

  19. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    International Nuclear Information System (INIS)

    Burcza, Anna; Gräf, Volker; Walz, Elke; Greiner, Ralf

    2015-01-01

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated

  20. Performance of juvenile mojarra supplied with feed containing varying levels of crude protein

    Directory of Open Access Journals (Sweden)

    Ricardo Henrique Bastos de Souza

    2016-04-01

    Full Text Available ABSTRACT The growth of the Brazilian aquaculture has stimulated the development of the productive chain of native species, including marine environment. The objective of this study was to evaluate the growth performance of juvenile mojarra fish (Diapterus rhombeus fed diets containing different concentrations of crude protein (32, 36, 40 and 44 g 100 g-1. The 80 juvenile mojarra (7.2±1.5 g were kept in 16 circular tanks (150 L. The study design used was completely randomized with four treatments and four repetitions. The fish were fed four times a day. At the end of the experiment (60 days the final weight, feed intake, weight gain (WG, feed:gain ratio (FGR, protein efficiency rate (PER, energy efficiency rate, specific growth, survival rate and, body composition were evaluated. It was verified significant effect of protein level on the WG, with the best value at the level of 38.20 g 100 g-1 of crude protein. For FGR, the best estimated value occurred with 38.06 g 100 g-1 of crude protein, similar to that reported for the PER (38.91 g 100 g-1. The other performance parameters and body composition were not influenced by crude protein levels. Diet crude protein concentrations between 38.06 and 38.91 g 100 g-1 provide the best performance indices for juvenile mojarra.

  1. Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Bastiaan A van den Berg

    Full Text Available Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

  2. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  3. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation.

    Science.gov (United States)

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N; Vainchenker, William; Solary, Eric; Giraudier, Stéphane

    2014-09-25

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. © 2014 by The American Society of Hematology.

  4. Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

    Directory of Open Access Journals (Sweden)

    Muscariello Lidia

    2009-02-01

    Full Text Available Abstract Background Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. Results We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa, which specifically binds to human fibronectin (Fn, an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. Conclusion Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying

  5. Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

    Science.gov (United States)

    Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

    2010-06-01

    Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

  6. Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves.

    Science.gov (United States)

    Kulkarni, Ketav P; Ramarathinam, Sri H; Friend, James; Yeo, Leslie; Purcell, Anthony W; Perlmutter, Patrick

    2010-06-21

    A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.

  7. Electron mobility on the surface of liquid Helium: influence of surface level atoms and depopulation of lowest subbands

    International Nuclear Information System (INIS)

    Grigoriev, P. D.; Dyugaev, A. M.; Lebedeva, E. V.

    2008-01-01

    The temperature dependence of electron mobility is examined. We calculate the contribution to the electron scattering rate from the surface level atoms (SLAs), proposed in [10]. This contribution is substantial at low temperatures T < 0.5, when the He vapor concentration is exponentially small. We also study the effect of depopulation of the lowest energy subband, which leads to an increase in the electron mobility at high temperature. The results explain certain long-standing discrepancies between the existing theory and experiment on electron mobility on the surface of liquid helium

  8. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  9. 2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

    Science.gov (United States)

    Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

    2015-12-01

    Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

  10. Multi-level machine learning prediction of protein–protein interactions in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Julian Zubek

    2015-07-01

    Full Text Available Accurate identification of protein–protein interactions (PPI is the key step in understanding proteins’ biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein–protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein–protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC. Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent.

  11. Level of C - reactive protein as an indicator for prognosis of premature uterine contractions.

    Science.gov (United States)

    Najat Nakishbandy, Bayar M; Barawi, Sabat A M

    2014-01-01

    high concentrations of maternal C-reactive protein have been associated with adverse pregnancy outcome, and premature uterine contraction may be predicted by elevated levels of C-reactive protein. This may ultimately be simple and cost-effective enough to introduce as a low-risk screening program. an observational case control study was performed from May 1st, 2010 to December 1st, 2010 at Maternity Teaching Hospital-Erbil/ Kurdistan Region/ Iraq. The sample size was (200) cases. Hundred of them were presented with premature uterine contractions at 24(+0)-36(+6) weeks. The other hundred were control group at same gestational ages. The level of C-reactive protein was determined in both groups and both groups were followed till delivery. (93) out of (100) women with premature uterine contractions had elevated level of C-Reactive protein and 91% delivered prematurely while in the control group only (9) out of (100) women had elevated level of C-reactive protein and only 8% of them delivered preterm. Differences were statistically highly significant. C-reactive protein can be used as a biomarker in prediction of premature delivery when it is associated with premature uterine contractions. As well it can be used as a screening test to detect cases that are at risk of premature delivery.

  12. Surface force analysis of molecular interfacial interactions of proteins and lipids with polymeric biomaterials

    International Nuclear Information System (INIS)

    Hamilton-Brown, P.; Griesser, H.J.; Meagher, L.

    2001-01-01

    Full text: Adverse biological responses to biomedical devices are often caused by the irreversible accumulation of biological deposits onto the surfaces of devices. Such deposits cause blocking of artificial blood vessels, fibrous encapsulation of soft tissue regenerative devices, 'fouling' of contact lenses, secondary cataracts on intraocular lenses, and other undesirable events that interfere with the intended functions of biomedical devices. The formation of deposits is triggered by an initial stage in which various proteins and lipids rapidly adsorb onto the synthetic material surface; further biological molecules and ultimately cellular entities (e.g., host cells, bacteria) then settle onto the initial adsorbed layer. Hence, to avoid or control the accumulation of biological deposits, molecular understanding is required of the initial adsorption processes. Such adsorption is caused by attractive interfacial forces, which we are characterising by the use of a novel method. In the present study, polymeric thin film coatings, polyethylene oxide (PEO), and polysaccharide coatings have been analysed in terms of their surface forces and the ensuing propensity for protein and lipid adsorption. Interfacial forces are measured using atomic force microscopy (AFM) with a colloid-modified tip in a liquid cell using solutions of physiological pH and ionic strength. The chemical composition and uniformity of the coatings was characterised by X-ray Photon Spectroscopy (XPS). For a polymeric solid coating, repulsive forces have been measured against a silica colloid probe, and the dominant surface force is electrostatic. For the highly hydrated, 'soft' PEO and polysaccharide coatings, on the other hand, steric/entropic forces are also significant and contribute to interfacial interactions with proteins and lipids. In one system we have observed a time dependence of the electrostatic surface potential, which affects interaction with charged proteins. Force measurements were

  13. 3D-SURFER 2.0: web platform for real-time search and characterization of protein surfaces.

    Science.gov (United States)

    Xiong, Yi; Esquivel-Rodriguez, Juan; Sael, Lee; Kihara, Daisuke

    2014-01-01

    The increasing number of uncharacterized protein structures necessitates the development of computational approaches for function annotation using the protein tertiary structures. Protein structure database search is the basis of any structure-based functional elucidation of proteins. 3D-SURFER is a web platform for real-time protein surface comparison of a given protein structure against the entire PDB using 3D Zernike descriptors. It can smoothly navigate the protein structure space in real-time from one query structure to another. A major new feature of Release 2.0 is the ability to compare the protein surface of a single chain, a single domain, or a single complex against databases of protein chains, domains, complexes, or a combination of all three in the latest PDB. Additionally, two types of protein structures can now be compared: all-atom-surface and backbone-atom-surface. The server can also accept a batch job for a large number of database searches. Pockets in protein surfaces can be identified by VisGrid and LIGSITE (csc) . The server is available at http://kiharalab.org/3d-surfer/.

  14. Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

    2014-02-01

    A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

  15. Harmful effect of protein difficiency on lipids, glucose, insulin and estradiol levels in female albino rats

    International Nuclear Information System (INIS)

    El-Mahdy, A.A.; El-Sherbiny, E.M.; Bayomi, M.M.

    2005-01-01

    The present study was undertaken to investigate the harmful effect of protein deficient diet on some biochemical activities in serum of female rats. Protein malnutrition is a well known socioeconomic problem in different parts of the world. Many studies were investigated on the biological parameters following protein malnutrition in human and experimental animals. Forty albino female rats were divided into 3 groups. The first group (10 rats) fed 18% protein diet and served as normal control and the other two groups, each contains 15 rats, fed 5% protein for 21 and 45 days, respectively, and served as malnourished groups. The results showed significant decrease in total body weight, serum glucose, insulin and estradiol levels in the third group as well as decrease in the total cholesterol, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol and triglycerides concentrations that compared to normal control rats

  16. Protein Consumption and the Elderly: What Is the Optimal Level of Intake?

    Directory of Open Access Journals (Sweden)

    Jamie I. Baum

    2016-06-01

    Full Text Available Maintaining independence, quality of life, and health is crucial for elderly adults. One of the major threats to living independently is the loss of muscle mass, strength, and function that progressively occurs with aging, known as sarcopenia. Several studies have identified protein (especially the essential amino acids as a key nutrient for muscle health in elderly adults. Elderly adults are less responsive to the anabolic stimulus of low doses of amino acid intake compared to younger individuals. However, this lack of responsiveness in elderly adults can be overcome with higher levels of protein (or essential amino acid consumption. The requirement for a larger dose of protein to generate responses in elderly adults similar to the responses in younger adults provides the support for a beneficial effect of increased protein in older populations. The purpose of this review is to present the current evidence related to dietary protein intake and muscle health in elderly adults.

  17. Sequence variations and protein expression levels of the two immune evasion proteins Gpm1 and Pra1 influence virulence of clinical Candida albicans isolates.

    Science.gov (United States)

    Luo, Shanshan; Hipler, Uta-Christina; Münzberg, Christin; Skerka, Christine; Zipfel, Peter F

    2015-01-01

    Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence.

  18. Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

    Science.gov (United States)

    Mallik, Saurav; Das, Smita; Kundu, Sudip

    2016-01-01

    Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.

  19. 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.

  20. Protein synthesis levels are increased in a subset of individuals with Fragile X syndrome

    DEFF Research Database (Denmark)

    Jacquemont, Sébastien; Pacini, Laura; Jønch, Aia E

    2018-01-01

    architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical...... severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS...... and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those...

  1. Load-dependent surface diffusion model for analyzing the kinetics of protein adsorption onto mesoporous materials.

    Science.gov (United States)

    Marbán, Gregorio; Ramírez-Montoya, Luis A; García, Héctor; Menéndez, J Ángel; Arenillas, Ana; Montes-Morán, Miguel A

    2018-02-01

    The adsorption of cytochrome c in water onto organic and carbon xerogels with narrow pore size distributions has been studied by carrying out transient and equilibrium batch adsorption experiments. It was found that equilibrium adsorption exhibits a quasi-Langmuirian behavior (a g coefficient in the Redlich-Peterson isotherms of over 0.95) involving the formation of a monolayer of cyt c with a depth of ∼4nm on the surface of all xerogels for a packing density of the protein inside the pores of 0.29gcm -3 . A load-dependent surface diffusion model (LDSDM) has been developed and numerically solved to fit the experimental kinetic adsorption curves. The results of the LDSDM show better fittings than the standard homogeneous surface diffusion model. The value of the external mass transfer coefficient obtained by numerical optimization confirms that the process is controlled by the intraparticle surface diffusion of cyt c. The surface diffusion coefficients decrease with increasing protein load down to zero for the maximum possible load. The decrease is steeper in the case of the xerogels with the smallest average pore diameter (∼15nm), the limit at which the zero-load diffusion coefficient of cyt c also begins to be negatively affected by interactions with the opposite wall of the pore. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Exposure of the Plasmodium falciparum clonally variant STEVOR proteins on the merozoite surface

    Directory of Open Access Journals (Sweden)

    Meri Seppo

    2011-03-01

    Full Text Available Abstract Background Plasmodium falciparum merozoites are free invasive forms that invade host erythrocytes in iterative cycles in the presence of different arms of the immune system. Variant antigens are known to play a role in immune evasion and several gene families coding for variant antigens have been identified in P. falciparum. However, none of them have been reported to be expressed on the surface of merozoites. Methods Flow cytometry, immunofluorescence microscopy, and immunoblotting assays were performed to assess surface exposure, membrane association and stage specific expression of the STEVOR family of variants proteins, respectively. Results Using a polyclonal antibody (anti-PFL2610w with a broad specificity towards different STEVOR variants, the STEVOR proteins were identified on the surface of non-permeabilized/non-fixed merozoites in flow cytometry assays. Anti-PFL2610w antibody showed that several STEVORs were expressed in the trophozoite stage of the parasite but only one variant was integrated into the merozoite membrane. Moreover, this antibody failed to identify STEVORs on the surface of the parent schizont infected erythrocytes (IE although they were readily identified when schizont IE were permeabilized. Conclusions These data suggest for a role for STEVOR in immune evasion by P. falciparum merozoites to allow successful invasion of erythrocytes. Additionally, the expression of STEVORs in the schizont stage may only represent a step in the biogenesis process of the merozoite surface coat.

  3. Genetically encoded pH sensor for tracking surface proteins through endocytosis.

    Science.gov (United States)

    Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D; Watkins, Simon C; Waggoner, Alan S; Bruchez, Marcel P

    2012-05-14

    Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Mosaic amino acid conservation in 3D-structures of surface protein and polymerase of hepatitis B virus

    NARCIS (Netherlands)

    van Hemert, Formijn J.; Zaaijer, Hans L.; Berkhout, Ben; Lukashov, Vladimir V.

    2008-01-01

    Surface protein and polymerase of hepatitis B virus provide a striking example of gene overlap. Inclusion of more coding constraints in the phylogenetic analysis forces the tree toward accepted topology. Three-dimensional protein modeling demonstrates that participation in local protein function

  5. Do infants with cow's milk protein allergy have inadequate levels of vitamin D?

    Science.gov (United States)

    Silva, Cristiane M; Silva, Silvia A da; Antunes, Margarida M de C; Silva, Gisélia Alves Pontes da; Sarinho, Emanuel Sávio Cavalcanti; Brandt, Katia G

    To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29ng/mL; p=0.041) and higher deficiency frequency (20.3% vs.8.2; p=0.049) than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p=0.002). Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p=0.972). Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  6. Do infants with cow's milk protein allergy have inadequate levels of vitamin D?

    Directory of Open Access Journals (Sweden)

    Cristiane M. Silva

    Full Text Available Abstract Objective: To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. Methods: This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30 ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Results: Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29 ng/mL; p = 0.041 and higher deficiency frequency (20.3% vs.8.2; p = 0.049 than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p = 0.002. Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p = 0.972. Conclusions: Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency.

  7. Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

    Science.gov (United States)

    Zhang, Xi

    2015-09-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

  9. Protein-protein networks construction and their relevance measurement based on multi-epitope-ligand-kartographie and gene ontology data of T-cell surface proteins for polymyositis.

    Science.gov (United States)

    Li, Fang-Zhen; Gao, Feng

    2012-08-01

    Polymyositis is an inflammatory myopathy characterized by muscle invasion of T-cells penetrating the basal lamina and displacing the plasma membrane of normal muscle fibers. In order to understand the different adhesive mechanisms at the T-cell surface, Schubert randomly selected 19 proteins expressed at the T-cell surface and studied them using MELK technique [4], among which 15 proteins are picked up for further study by us. Two types of functional similarity networks are constructed for these proteins. The first type is MELK similarity network, which is constructed based on their MELK data by using the McNemar's test [24]. The second type is GO similarity network, which is constructed based on their GO annotation data by using the RSS method to measuring functional similarity. Then the subset surprisology theory is employed to measure the degree of similarity between two networks. Our computing results show that these two types of networks are high related. This conclusion added new values on MELK technique and expanded its applications greatly.

  10. Analysis of Pseudomonas aeruginosa cell envelope proteome by capture of surface-exposed proteins on activated magnetic nanoparticles.

    Directory of Open Access Journals (Sweden)

    Davide Vecchietti

    Full Text Available We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.

  11. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

    Science.gov (United States)

    Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

    2012-01-01

    We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria. PMID:23226459

  12. Urea metabolism in buffalo calves fed on rations containing two levels of crude protein

    International Nuclear Information System (INIS)

    Verma, D.N.; Singh, U.B.; Lal, M.; Varma, A.; Ranjhan, S.K.

    1974-01-01

    Urea entry rates into the body pools of Murrah Buffalo calves have been estimated using a single injection isotope dilution technique using 14 C-urea. The animals were fed two levels of crude proteins, namely, 13 percent lower and 19 percent higher than N.R.C. recommendations. Results show that the recycling of urea is significantly better in animals given low crude protein contents. (M.G.B.)

  13. Effects of dietary protein levels on length-weight relationships and ...

    African Journals Online (AJOL)

    Feeding trial involving different protein levels on length–weight relationships and condition factor of Clarias gariepinus was conducted in floating hapa system. Fingerlings (average weight, 4.50± 0.01g and average length, 8.0±0.2 cm) were randomly stocked at 20 fish/1m3. Five diets with crude protein: 40.0, 42.5, 45.0, 47.5 ...

  14. Levels of surface contamination with radioactive materials at workplaces of nuclear research centre at Rez

    International Nuclear Information System (INIS)

    Hoelgye, Z.; Nemcova, I.; Kasikova, M.; Popper, J.; Chysky, J.

    1983-01-01

    A hygiene supervision unit at workplaces of the nuclear Research Institute in Rez monitored on a long-term basis surface contamination with radioactive substances. Surface contamination was found at workplaces with open sources. Of the 4343 monitored places action levels were only exceeded in 13 cases. The obtained data were used for typifying workplaces with the highest level of surface contamination, to determine in certain instances the mechanism of the escape of radioactive substances from insulating facilities and to determine the rate of the spread of the radioactive substance into adjacent non-active workplaces. (author)

  15. Behavior of Rydberg atoms at surfaces: energy level shifts and ionization

    Energy Technology Data Exchange (ETDEWEB)

    Dunning, F.B. E-mail: fbd@rice.edu; Dunham, H.R.; Oubre, C.; Nordlander, P

    2003-04-01

    The ionization of xenon atoms excited to the extreme red and blue states in high-lying Xe(n) Stark manifolds at a metal surface is investigated. The data show that, despite their very different initial spatial characteristics, the extreme members of a given Stark manifold ionize at similar atom/surface separations. This is explained, with the aid of complex scaling calculations, in terms of the strong perturbations in the energies and structure of the atomic states induced by the presence of the surface which lead to avoided crossings between neighboring levels as the surface is approached.

  16. Behavior of Rydberg atoms at surfaces: energy level shifts and ionization

    CERN Document Server

    Dunning, F B; Oubre, C D; Nordlander, P

    2003-01-01

    The ionization of xenon atoms excited to the extreme red and blue states in high-lying Xe(n) Stark manifolds at a metal surface is investigated. The data show that, despite their very different initial spatial characteristics, the extreme members of a given Stark manifold ionize at similar atom/surface separations. This is explained, with the aid of complex scaling calculations, in terms of the strong perturbations in the energies and structure of the atomic states induced by the presence of the surface which lead to avoided crossings between neighboring levels as the surface is approached.

  17. Molecular surface representation using 3D Zernike descriptors for protein shape comparison and docking.

    Science.gov (United States)

    Kihara, Daisuke; Sael, Lee; Chikhi, Rayan; Esquivel-Rodriguez, Juan

    2011-09-01

    The tertiary structures of proteins have been solved in an increasing pace in recent years. To capitalize the enormous efforts paid for accumulating the structure data, efficient and effective computational methods need to be developed for comparing, searching, and investigating interactions of protein structures. We introduce the 3D Zernike descriptor (3DZD), an emerging technique to describe molecular surfaces. The 3DZD is a series expansion of mathematical three-dimensional function, and thus a tertiary structure is represented compactly by a vector of coefficients of terms in the series. A strong advantage of the 3DZD is that it is invariant to rotation of target object to be represented. These two characteristics of the 3DZD allow rapid comparison of surface shapes, which is sufficient for real-time structure database screening. In this article, we review various applications of the 3DZD, which have been recently proposed.

  18. Interaction betwen Lead and Bone Protein to Affect Bone Calcium Level Using UV-Vis Spectroscopy

    Science.gov (United States)

    Noor, Z.; Azharuddin, A.; Aflanie, I.; Kania, N.; Suhartono, E.

    2018-05-01

    This present study aim to evaluate the interactions between lead (Pb) and with bone protein by UV-Vis approach. In addition, this prsent study also aim to investigate the effect of Pb on bone calcium (Ca) level. The present study was a true experimental study design to examine the impact of Pb exposure in bone of male rats (Rattus novergicus). The study involved 5 groups, P1 was the control group, while the other (P2-P5) were the case group with exposure of Pb in different concentration within 4 weeks. At the end of the exposure, the interaction between Pb and protein was determined using UV-Vis spectrophotometric method, and the Ca level was determined using permanganometric method. The results shows that that there is an interaction between Pb and bone protein. The result also shows that the value of the binding constant of Protein-Pb is 32.71. It means Pb have an high affinity to bind with bone protein, which promote a further reaction to induced the release of bone Ca from the bone protein. In conclusion, this present study found an obvious relationship between Pb and bone protein which promote a further reaction to increase the releasing of bone calcium.

  19. Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

    DEFF Research Database (Denmark)

    Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

    2008-01-01

    diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

  20. Serum eosinophil cationic protein levels can be useful for predicting acute exacerbation of asthma

    Directory of Open Access Journals (Sweden)

    Mitsuhiro Kamimura

    1998-01-01

    Full Text Available We report on a case in which five consecutive exacerbations of asthma were monitored by following serum eosinophil cationic protein (ECP levels. The serum ECP level correlated well with each exacerbation and tended to increase even before the exacerbations of asthma became apparent. This case shows that serum levels of ECP can be useful markers of disease activity and may also be predictive markers for acute exacerbation.

  1. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system.

    Directory of Open Access Journals (Sweden)

    Rong Gao

    2013-10-01

    Full Text Available Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS. In response to phosphate (Pi-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural

  2. Protein analysis in dissolved organic matter: What proteins from organic debris, soil leachate and surface water can tell us - a perspective

    Directory of Open Access Journals (Sweden)

    W. X. Schulze

    2005-01-01

    Full Text Available Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM, this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1 the identification of phylogenetic groups contributing to the environmental protein pool, and (2 identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from decomposing plant material and DOM of soil leachates and surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from litter protein extracts, leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  3. Ground level environmental protein concentrations in various ecuadorian environments: potential uses of aerosolized protein for ecological research

    Science.gov (United States)

    Staton, Sarah J.R.; Woodward, Andrea; Castillo, Josemar A.; Swing, Kelly; Hayes, Mark A.

    2014-01-01

    Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m3, highly altered 0.07 ± 0.05 μg/m3, regrowth transitional 0.17 ± 0.06 μg/m3, and suburban deforested 0.09 ± 0.04 μg/m3. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m3 and wet season 0.10 ± 0.04 μg/m3), environmental fragmentation (buffered 0.19 ± 0.06 μg/m3 and edge 0.15 ± 0.06 μg/m3), and sampling height (ground level 0.32 ± 0.09 μg/m3 and 10 m 0.24 ± 0.04 μg/m3) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r2 = 0.801), net primary production (NPP) (r2 = 0.827), leaf area index (LAI) (r2 = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated

  4. Sub-nanoscale surface ruggedness provides a water-tight seal for exposed regions in soluble protein structure.

    Directory of Open Access Journals (Sweden)

    Erica Schulz

    2010-09-01

    Full Text Available Soluble proteins must maintain backbone hydrogen bonds (BHBs water-tight to ensure structural integrity. This protection is often achieved by burying the BHBs or wrapping them through intermolecular associations. On the other hand, water has low coordination resilience, with loss of hydrogen-bonding partnerships carrying significant thermodynamic cost. Thus, a core problem in structural biology is whether natural design actually exploits the water coordination stiffness to seal the backbone in regions that are exposed to the solvent. This work explores the molecular design features that make this type of seal operative, focusing on the side-chain arrangements that shield the protein backbone. We show that an efficient sealing is achieved by adapting the sub-nanoscale surface topography to the stringency of water coordination: an exposed BHB may be kept dry if the local concave curvature is small enough to impede formation of the coordination shell of a penetrating water molecule. Examination of an exhaustive database of uncomplexed proteins reveals that exposed BHBs invariably occur within such sub-nanoscale cavities in native folds, while this level of local ruggedness is absent in other regions. By contrast, BHB exposure in misfolded proteins occurs with larger local curvature promoting backbone hydration and consequently, structure disruption. These findings unravel physical constraints fitting a spatially dependent least-action for water coordination, introduce a molecular design concept, and herald the advent of water-tight peptide-based materials with sufficient backbone exposure to remain flexible.

  5. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. ProtPOS: a python package for the prediction of protein preferred orientation on a surface.

    Science.gov (United States)

    Ngai, Jimmy C F; Mak, Pui-In; Siu, Shirley W I

    2016-08-15

    Atomistic molecular dynamics simulation is a promising technique to investigate the energetics and dynamics in the protein-surface adsorption process which is of high relevance to modern biotechnological applications. To increase the chance of success in simulating the adsorption process, favorable orientations of the protein at the surface must be determined. Here, we present ProtPOS which is a lightweight and easy-to-use python package that can predict low-energy protein orientations on a surface of interest. It combines a fast conformational sampling algorithm with the energy calculation of GROMACS. The advantage of ProtPOS is it allows users to select any force fields suitable for the system at hand and provide structural output readily available for further simulation studies. ProtPOS is freely available for academic and non-profit uses at http://cbbio.cis.umac.mo/software/protpos Supplementary data are available at Bioinformatics online. shirleysiu@umac.mo. © The Author 2016. Published by Oxford University Press.

  7. 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.

  8. Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection

    Science.gov (United States)

    Bakhmachuk, A.; Gorbatiuk, O.; Rachkov, A.; Dons'koi, B.; Khristosenko, R.; Ushenin, I.; Peshkova, V.; Soldatkin, A.

    2017-02-01

    The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 μg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement ( r 2 = 0.97) with the given concentration of IgG.

  9. Bone Morphogenetic Protein Coating on Titanium Implant Surface: a Systematic Review

    Directory of Open Access Journals (Sweden)

    Haim Haimov

    2017-06-01

    Full Text Available Objectives: The purpose of the study is to systematically review the osseointegration process improvement by bone morphogenetic protein coating on titanium implant surface. Material and Methods: An electronic literature search was conducted through the MEDLINE (PubMed and EMBASE databases. The search was restricted for articles published during the last 10 years from October 2006 to September 2016 and articles were limited to English language. Results: A total of 41 articles were reviewed, and 8 of the most relevant articles that are suitable to the criteria were selected. Articles were analysed regarding concentration of bone morphogenetic protein (BMP, delivery systems, adverse reactions and the influence of the BMP on the bone and peri-implant surface in vivo. Finally, the present data included 340 implants and 236 models. Conclusions: It’s clearly shown from most of the examined studies that bone morphogenetic protein increases bone regeneration. Further studies should be done in order to induce and sustain bone formation activity. Osteogenic agent should be gradually liberated and not rapidly released with priority to three-dimension reservoir (incorporated titanium implant surface in order to avoid following severe side effects: inflammation, bleeding, haematoma, oedema, erythema, and graft failure.

  10. Fabrication of Surface Protein-Imprinted Nanoparticles Using a Metal Chelating Monomer via Aqueous Precipitation Polymerization.

    Science.gov (United States)

    Li, Wei; Sun, Yan; Yang, Chongchong; Yan, Xianming; Guo, Hao; Fu, Guoqi

    2015-12-16

    Molecular imprinting is a promising way for constructing artificial protein recognition materials, but it has been challenged by difficulties such as restricted biomacromolecule transfer in the cross-linked polymer networks, and reduced template-monomer interactions that are due to the required aqueous media. Herein, we propose a strategy for imprinting of histidine (His)-exposed proteins by combining previous approaches such as surface imprinting over nanostructures, utilization of metal coordination interactions, and adoption of aqueous precipitation polymerization capable of forming reversible physical crosslinks. With lysozyme as a model template bearing His residues, imprinted polymer nanoshells were grafted over vinyl-modified nanoparticles by aqueous precipitation copolymerization of a Cu(2+) chelating monomer with a temperature-responsive monomer carried out at 37 °C, above the volume phase-transition temperature (VPTT) of the final copolymer. The imprinted nanoshells showed significant temperature sensitivity and the template removal could be facilitated by swelling of the imprinted layers at 4 °C, below the VPTT. The resultant core-shell imprinted nanoparticles exhibited strikingly high rebinding selectivity against a variety of nontemplate proteins. An imprinting factor up to 22.7 was achieved, which is among the best values reported for protein imprinting, and a rather high specific binding capacity of 67.3 mg/g was obtained. Moreover, this approach was successfully extended to preliminary imprinting of hemoglobin, another protein with accessible His. Therefore, it may be a versatile method for fabrication of high-performance surface-imprinted nanoparticles toward His-exposed proteins.

  11. Noise genetics: inferring protein function by correlating phenotype with protein levels and localization in individual human cells.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    2014-03-01

    Full Text Available To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.

  12. Biosensing for the Environment and Defence: Aqueous Uranyl Detection Using Bacterial Surface Layer Proteins

    Directory of Open Access Journals (Sweden)

    David J.R. Conroy

    2010-05-01

    Full Text Available The fabrication of novel uranyl (UO22+ binding protein based sensors is reported. The new biosensor responds to picomolar levels of aqueous uranyl ions within minutes using Lysinibacillus sphaericus JG-A12 S-layer protein tethered to gold electrodes. In comparison to traditional self assembled monolayer based biosensors the porous bioconjugated layer gave greater stability, longer electrode life span and a denser protein layer. Biosensors responded specifically to UO22+ ions and showed minor interference from Ni2+, Cs+, Cd2+ and Co2+. Chemical modification of JG-A12 protein phosphate and carboxyl groups prevented UO22+ binding, showing that both moieties are involved in the recognition to UO22+.

  13. The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

    Science.gov (United States)

    Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

    2009-01-01

    Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

  14. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    Science.gov (United States)

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  15. Durable grafting of silkworm pupa protein onto the surface of polyethylene terephthalate fibers

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jianfeng, E-mail: 584884673@qq.com [College of Textiles & Garments, Southwest University, Chongqing 400716 (China); Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, 400716 (China); Zheng, Dandan, E-mail: 183737543@qq.com [College of Textiles & Garments, Southwest University, Chongqing 400716 (China); Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, 400716 (China); Zhang, Fengxiu, E-mail: zhangfx656472@sina.com.cn [School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Zhang, Guangxian, E-mail: zgx656472@sina.com [College of Textiles & Garments, Southwest University, Chongqing 400716 (China); Chongqing Engineering Research Center of Biomaterial Fiber and Modern Textile, 400716 (China)

    2016-12-01

    In this paper, reactive –NH{sub 2} groups (8.36 × 10{sup −6} mol/g fabric) were introduced to the surface of polyethylene terephthalate (PET) fabrics by a nitration and reduction method, and epoxy groups were introduced to silkworm pupa protein (SPP) by reaction with epoxy chloropropane. PET-SPP composite fabrics were then prepared by reaction of these two precursors. The results showed that the SPP was firmly grafted onto the PET fabric surface and that the hydrophilicity of the fabric was markedly improved by the grafting of SPP. SEM images revealed a layer of substance covering the surface of the PET fibers, and XPS investigation showed that the nitrogen content of the PET-SPP fabric was higher than that of the original PET fabric (2.32% vs 0%). ATR-FTIR adsorption bands at 1653 and 1543 cm{sup −1} suggested the successful grafting of SPP onto the PET fabric surface. The DSC and TG of the PET fibers demonstrated that the thermal stability of the original PET fibers was maintained well by the SPP-grafted PET fibers. The breaking strength, bending rigidity, air permeability, and crease recovery angle of the original PET fabric were also retained by the SPP-grafted PET fabric. - Highlights: • Reactive –NH{sub 2} groups were introduced to PET fibers by nitration and reduction method. • Reactive epoxy groups were introduced to silkworm pupa protein by reacting with epoxy chloropropane. • The silkworm pupa protein could be grafted firmly on the PET fabric surface through covalent bond. • The skin-friendly property and hydrophilicity of PET-SPP fabric were improved greatly. • The wearability of PET-SPP composite fabric kept well.

  16. Elevated levels of the mismatch repair protein PMS2 are associated with prostate cancer.

    Science.gov (United States)

    Norris, Alixanna M; Woodruff, R D; D'Agostino, Ralph B; Clodfelter, Jill E; Scarpinato, Karin Drotschmann

    2007-02-01

    Defects in mismatch repair (MMR) proteins have been identified in various types of cancer. However, an association with prostate cancer has been controversial. Defective MMR results in genome instability with detrimental consequences that significantly contribute to tumorigenesis. This study determined alterations in key MMR protein levels in prostate cancer with the goal to identify prognostic markers. Prostatectomy samples were immunohistochemically stained and the relative presence or absence of key proteins MSH2, MLH1, and PMS2 determined. Cancer tissue of distinct grades was compared with the normal surrounding tissue. Microsatellite instability (MSI) in altered tissues was determined according to NCI guidelines. In contrast to reports that associate a lack of individual MMR proteins with tumorigenesis, a significant increase in PMS2 levels was identified in PIN lesions and prostate cancer tissue. This elevation in PMS2 was independent of changes in levels in its heterodimeric partner, MLH1. Prostate tumors with elevated levels of PMS2 were genetically unstable, which was corrected by MLH1 co-elevation. This is the first documentation of detrimental consequences associated with the increase in a MMR protein in human cancer. This study recognizes PMS2 elevation as a prognostic marker in pre-neoplastic and prostate cancer lesions. This result has significant implications for future diagnostic and treatment measures. (c) 2006 Wiley-Liss, Inc.

  17. Molecular-level chemistry of model single-crystal oxide surfaces with model halogenated compounds

    Science.gov (United States)

    Adib, Kaveh

    Synchrotron-based X-ray photoelectron spectroscopy (XPS), temperature-programmed desorption (TPD) and low energy electron diffraction (LEED) have been used to investigate, at a molecular level, the chemistry of different terminations of single crystal iron-oxide surfaces with probe molecules (CCl4 and D2O). Comparisons of the reactivity of these surfaces towards CCl4, indicate that the presence of an uncapped surface Fe cation (strong Lewis acid site) and an adjacent oxygen site capped by that cation can effect the C-Cl bond cleavage in CCl4, resulting in dissociatively adsorbed Cl-adatoms and carbon-containing fragments. If in addition to these sites, an uncapped surface oxygen (Lewis base) site is also available, the carbon-containing moiety can then move that site, coordinate itself with that uncapped oxygen, and stabilize itself. At a later step, the carbon-containing fragment may form a strong covalent bond with the uncapped oxygen and may even abstract that surface oxygen. On the other hand, if an uncapped oxygen is not available to stabilize the carbon-containing fragment, the surface coordination will not occur and upon the subsequent thermal annealing of the surface the Cl-adatoms and the carbon-containing fragments will recombine and desorb as CCl4. Finally, the presence of surface deuteroxyls blocking the strong Lewis acid and base sites of the reactive surface, passivates this surface. Such a deuteroxylated surface will be unreactive towards CCl 4. Such a molecular level understanding of the surface chemistry of metal-oxides will have applications in the areas of selective catalysis, including environmental catalysis, and chemical sensor technology.

  18. Monitoring sea level and sea surface temperature trends from ERS satellites

    DEFF Research Database (Denmark)

    Andersen, Ole Baltazar; Knudsen, Per; Beckley, B.

    2002-01-01

    Data from the two ESA satellites ERS-1 and ERS-2 are used in global and regional analysis of sea level and sea surface temperature trends over the last, 7.8 years. T he ERS satellites and in the future the ENVISAT satellite provide unique opportunity for monitoring both changes in sea level and sea...

  19. Temporal stability of naturally acquired immunity to Merozoite Surface Protein-1 in Kenyan Adults

    Directory of Open Access Journals (Sweden)

    Crabb Brendan S

    2009-07-01

    Full Text Available Abstract Background Naturally acquired immunity to blood-stage Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens. Methods Blood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-142 was determined by IFN-γ ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-119 invasion inhibition antibodies (IIA to the E-TSR (3D7 allele and growth inhibitory activity (GIA. The haplotype of MSP-119 alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months. Results MSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-γ responses and GIA. MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 E-TSR haplotype. Conclusion Variation in the stability of cellular and humoral immune responses to P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.

  20. 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...

  1. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    Science.gov (United States)

    Pinna, C; Stefanelli, C; Biagi, G

    2014-12-01

    The aim of the present study was to evaluate in vitro the effect of some prebiotic substances and 2 dietary protein levels on the composition and activity of feline fecal microbiota. Two in vitro studies were conducted. First, 6 nondigestible oligosaccharides were studied; treatments were control diet (CTRL), gluconic acid (GA), carrot fiber (CF), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactitol (LAC), and pectins from citrus fruit (PEC). Substrates were added to feline fecal cultures at 2 g/L for 24 h incubation. Compared with the CTRL, ammonia had been reduced (Pmicrobiota and that high dietary protein levels in a cat's diet can have negative effects on the animal intestinal environment.

  2. Synthetic peptides derived from salivary proteins and the control of surface charge densities of dental surfaces improve the inhibition of dental calculus formation.

    Science.gov (United States)

    Grohe, Bernd

    2017-08-01

    Peptides descended from the salivary proteins statherin and histatin were recently identified in saliva and the acquired enamel pellicle (AEP), a proteomic layer coated on enamel. In particular, the statherin phosphopeptide DpSpSEEKFLR (DSS) was found to adsorb to enamel-like hydroxyapatite and inhibit plaque-related crystal formation. To determine the mechanism of these processes, we studied peptide-crystal interactions based on the sequences DSS and RKFHEKHHSHRGYR (RKF). The latter is a basic histatin sequence showing antimicrobial effects. To initiate crystallization we used calcium oxalate monohydrate (COM), a rather secondary phase in the oral environment, however highly amenable to experimental analyses of nucleation and growth processes. Using electron microscopy we found that the peptides DSS, DSS-RKF and DSS-DSS all inhibit crystal formation; with DSS-DSS showing the strongest effects while RKF showed no effect. In addition, using either enamel-like or mica substrates, we found that the ratio of the substrate's surface charge densities was directly correlated with the ratio of COM nucleation rates on theses surfaces. The findings suggest that mineralization processes on enamel/AEP-films are controllable by the degree of peptide phosphorylation/acidity and the level of the enamel surface charge density. Both parameters can, when well adjusted, help to overcome periodontal disease and dental calculus formation. In addition, the presence of antimicrobial RKF will reduce the buildup of bacterial plaque. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface.

    Science.gov (United States)

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2017-04-01

    Renal proximal tubule epithelial cells (PTECs) are known to reabsorb salts and small plasma proteins filtered through Bowman's capsule. Following acute kidney injury, PTECs assume some characteristics of hepatocytes in producing various plasma proteins. We now demonstrate that even at a resting state, a PTEC cell line, HK2 expresses mRNAs for and synthesizes and secretes plasma proteins in a complex with complement C3, an α 2 -macroglobulin family chaperone, including albumin, transferrin, α 1 -antitrypsin, α 1 -antichymotrypsin, α 2 -HS-glycoprotein, ceruloplasmin, haptoglobin, C1-inhibitor, secreted phosphoprotein-24, and insulin-like growth factor-1. When grown on transwell inserts, HK2 cells predominantly secrete (∼90%) plasma proteins into the apical side and a smaller fraction into the basolateral side as determined by ELISA assays. When cultured in the presence of exogenous cytokines such as IL1β, IL6, TNFα, BMP2, or TGFβ1, HK2 cell mRNA expressions for plasma proteins were variably affected whereas basolateral secretions were elevated to or in excess of those of the apical level. In addition, HK2 cells produce proTGFβ1 with its intact N-terminal latency associated peptide and latent-TGF-β-binding proteins. The complex cannot be dissociated under conditions of SDS, heating, and electrophoresis. Moreover, HK2 cells maintain their ability to quickly uptake exogenously added serum proteins from the culture medium, as if they are recognized differently by the endocytic receptors. These results provide new insight into the hepatization of PTECs. In addition to their unique uptake of plasma proteins and salts from the filtrate, they are a source of urinary proteins under normal conditions as wells as in chronic and acute kidney diseases. J. Cell. Biochem. 118: 924-933, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    International Nuclear Information System (INIS)

    Lu Yan; Yan Changling; Wang Xuejing; Wang Gongke

    2009-01-01

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

  5. Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

    Science.gov (United States)

    Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

    2016-06-07

    Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

  6. Phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma

    International Nuclear Information System (INIS)

    Low, M.G.; Prasad, A.R.S.

    1988-01-01

    An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or no activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only 3 H-labeled product when this enzyme hydrolyzed [ 3 H]myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca 2 =-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosylphosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo

  7. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  8. Merozoite surface protein-1 genetic diversity in Plasmodium malariae and Plasmodium brasilianum from Brazil.

    Science.gov (United States)

    Guimarães, Lilian O; Wunderlich, Gerhard; Alves, João M P; Bueno, Marina G; Röhe, Fabio; Catão-Dias, José L; Neves, Amanda; Malafronte, Rosely S; Curado, Izilda; Domingues, Wilson; Kirchgatter, Karin

    2015-11-16

    The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites. Except for fragment 4, sequences from all other fragments consisted of unpublished sequences. The most polymorphic gene region was fragment 2, and in samples where this region lacks polymorphism, all other regions are also identical. The low variability of the P. malariae msp1 sequences of these isolates and the identification of the same haplotype in those collected many years apart at different locations is compatible with a low transmission rate. We also found greater diversity among P. brasilianum isolates compared with P. malariae ones. Lastly, the sequences were segregated according to their geographic origins and hosts, showing a strong genetic and geographic structure. Our data

  9. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    Science.gov (United States)

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

  10. Accessible surface area of proteins from purely sequence information and the importance of global features

    Science.gov (United States)

    Faraggi, Eshel; Zhou, Yaoqi; Kloczkowski, Andrzej

    2014-03-01

    We present a new approach for predicting the accessible surface area of proteins. The novelty of this approach lies in not using residue mutation profiles generated by multiple sequence alignments as descriptive inputs. Rather, sequential window information and the global monomer and dimer compositions of the chain are used. We find that much of the lost accuracy due to the elimination of evolutionary information is recouped by the use of global features. Furthermore, this new predictor produces similar results for proteins with or without sequence homologs deposited in the Protein Data Bank, and hence shows generalizability. Finally, these predictions are obtained in a small fraction (1/1000) of the time required to run mutation profile based prediction. All these factors indicate the possible usability of this work in de-novo protein structure prediction and in de-novo protein design using iterative searches. Funded in part by the financial support of the National Institutes of Health through Grants R01GM072014 and R01GM073095, and the National Science Foundation through Grant NSF MCB 1071785.

  11. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    Science.gov (United States)

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2014-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

  12. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...... about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic...

  13. Surface core level shifts of clean and oxygen covered Ir(111)

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, M; Cassese, D; Cavallin, A; Comin, R; Orlando, F; Postregna, L [Universita degli Studi di Trieste, Via A Valerio 2, 34127, Trieste (Italy); Golfetto, E; Baraldi, A [Dipartimento di Fisica e CENMAT, Universita degli Studi di Trieste, Via A Valerio 2, 34127, Trieste (Italy); Lizzit, S [Sincrotrone Trieste S.C.p.A., S.S. 14 Km 163.5, 34012 Trieste (Italy)], E-mail: alessandro.baraldi@elettra.trieste.it

    2009-06-15

    We present the results of high resolution core level photoelectron spectroscopy employed to investigate the electronic structure of clean and oxygen covered Ir(111) surface. Ir 4f{sub 7/2} core level spectra are shown to be very sensitive to the local atomic environment. For the clean surface we detected two distinct components shifted by 550 meV, originated by surface and bulk atoms. The larger Gaussian width of the bulk component is explained as due to experimentally unresolved subsurface components. In order to determine the relevance of the phonon contribution we examined the thermal behaviour of the core level lineshape using the Hedin-Rosengren theory. From the phonon-induced spectral broadening we found the Debye temperature of bulk and surface atoms to be 298 and 181 K, respectively, which confirms the softening of the vibrational modes at the surface. Oxygen adsorption leads to the appearance of new surface core level components at -200 meV and +230 meV, which are interpreted as due to first-layer Ir atoms differently coordinated with oxygen. The coverage dependence of these components demonstrates that the oxygen saturation corresponds to 0.38 ML, in good agreement with recent density functional theory calculations.

  14. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The effects of maternal total protein, albumin and hemoglobin levels on birth weight

    Directory of Open Access Journals (Sweden)

    Berna Haliloglu

    2007-12-01

    Full Text Available OBJECTIVE: The present study was designed to investigate the influence of third trimester maternal total protein, albumin, hemoglobin levels on birth weight.\tMATERIAL-METHOD: Between January 2005 and July 2005, 750 pregnant women applied for delivery at Zeynep Kamil Women’s and Children Education and Research Hospital at 37-40 week’s gestation were examined. Maternal total protein, albumin and hemoglobin levels were measured. Data included maternal age, gravidity, parity, gestational age, birth weight, gender, presence of iron supplementation and its duration.\tRESULTS: The birth weight was significantly higher in anemic and hypoproteinemic groups compared those with normal levels. After adjusting for counfounding factors, significance of both findings lost. The cases received iron supplementation had infants with higher birth weight, however, it was not statistically significant (p: 0.055. A significant positive relation was observed between birth weight and maternal age, gravidity, parity and gestational age. No relation found between maternal total protein, albumin, hemoglobin levels and birth weight.\tCONCLUSION: The last trimester maternal total protein, albumin, hemoglobin levels seem not to be a determining factor on infant's birth weight.

  16. Research on near-surface disposal of very low level radioactive waste

    International Nuclear Information System (INIS)

    Wang Shaowei; Yue Huiguo; Hou Jie; Chen Haiying; Zuo Rui; Wang Jinsheng

    2012-01-01

    Radioactive waste disposal is one of the most sensitive environmental problems to control and solve. As the arriving of decommissioning of early period nuclear facilities in China, large amounts of very low level radioactive waste will be produced inevitably. The domestic and abroad definitions about very low level radioactive waste and its disposal were introduced, and then siting principles of near-surface disposal of very low level radioactive waste were discussed. The near- surface disposal siting methods of very low level radioactive waste were analyzed from natural and geographical conditions assessment, geological conditions analysis, hydrogeological conditions analysis, geological hazard assessment and radioactive background investigation; the near-surface disposal sites'natural barriers of very low level radioactive waste were analyzed from the crustal structure and physico-chemical characteristics, the dynamics characteristics of groundwater, the radionuclide adsorption characteristics of natural barriers and so on; the near-surface disposal sites' engineered barriers of very low level radioactive waste were analyzed from the repository design, the repository barrier materials selection and so on. Finally, the improving direction of very low level radioactive waste disposal was proposed. (authors)

  17. ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family

    International Nuclear Information System (INIS)

    Caesar, Joseph J. E.; Johnson, Steven; Kraiczy, Peter; Lea, Susan M.

    2013-01-01

    The structure of ErpC, a member of the complement regulator-acquiring surface protein family from B. burgdorferi, has been solved, providing insights into the strategies of complement evasion by this zoonotic bacterium and suggesting a common architecture for other members of this protein family. Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts

  18. 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

  19. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

    Science.gov (United States)

    Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

    2011-05-26

    Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we dem