Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge.

Science.gov (United States)

Ma, Chunli; Zhang, Lili; Gao, Mingyang; Ma, Dexing

2017-07-01

Two novel plasmids pTX8048-SP-Δ3-1E and pTX8048-SP-NAΔ3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-Δ3-1E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAΔ3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1E-specific serum IgG, increased high proportion of CD4 + and CD8α + cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Science.gov (United States)

Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

2017-12-01

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

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.

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

Science.gov (United States)

Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

2018-02-28

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application

Science.gov (United States)

2018-01-01

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids. PMID:29503972

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.

Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis.

Science.gov (United States)

Bruneau, J M; Magnin, T; Tagat, E; Legrand, R; Bernard, M; Diaquin, M; Fudali, C; Latgé, J P

2001-08-01

Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W. A., Diaquin M., Popolo L., Hartland R. P., Latgé J.-P, J. Biol. Chem. 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane. To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A. fumigatus and the protein data were matched with the yeast genomic data. GPI-anchored proteins of A. fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis. They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing. Nine GPI-anchored proteins were identified in A. fumigatus. Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis. In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A. fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins. These results suggest that GPI-anchored proteins identified in this study are involved in A. fumigatus cell wall organization.

Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.

Science.gov (United States)

Bowman, Shaun M; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J

2006-03-01

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

NARCIS (Netherlands)

Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

2015-01-01

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

NARCIS (Netherlands)

Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

2015-01-01

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

Science.gov (United States)

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibers in B. subtilis Biofilms

Science.gov (United States)

Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2011-01-01

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibers, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibers. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibers to the cell wall and to assemble TasA into fibers. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibers can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibers. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibers that are not anchored to the cell. PMID:21477127

An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.

Science.gov (United States)

Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2011-06-01

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell. © 2011 Blackwell Publishing Ltd.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Science.gov (United States)

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory, Variability, and Virulence

Science.gov (United States)

Rittenour, William R.; Harris, Steven D.

2013-01-01

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers. PMID:24312325

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

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

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.

Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis.

Science.gov (United States)

Gillmor, C Stewart; Lukowitz, Wolfgang; Brininstool, Ginger; Sedbrook, John C; Hamann, Thorsten; Poindexter, Patricia; Somerville, Chris

2005-04-01

Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth.

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

International Nuclear Information System (INIS)

Mayor, S.; Menon, A.K.; Cross, G.A.

1990-01-01

A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with [3H]palmitic acid and [3H]myristic acid show that [3H]palmitic acid specifically labels the inositol residue in P3 while [3H]myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors

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.

Anomalous carrier life-time relaxation mediated by head group interaction in surface anchored MnSe quantum dots conjugated with albumin proteins

Energy Technology Data Exchange (ETDEWEB)

Sarma, Runjun; Mohanta, Dambarudhar, E-mail: best@tezu.ernet.in

2017-02-01

We report on the radiative emission decay dynamics of a less known, γ-phase manganese selenide quantum dot system (MnSe QDs) subjected to bio-functionalization. A short-ligand thioglycolic acid (TGA), and a long-chain sodium dodecyl sulfate (SDS) surfactants were used as surface anchors prior bioconjugation with albumin proteins (BSA). Time resolved photoluminescence (TR-PL) spectra of the QDs have revealed bi-exponential decay trends with the fast (τ{sub 1}) and slow (τ{sub 2}) decay parameters assigned to the core state recombination and surface trapped excitons; respectively. The average lifetime (τ{sub avg}) was found to get shortened from a value of ∼0.87 ns–0.72 ns in unconjugated and BSA conjugated MnSe-TGA QDs; respectively. Conversely, MnSe-SDS QDs with BSA conjugation exhibited nearly four-fold enhancement of τ{sub avg} with respect to its unconjugated counterpart. Moreover, a considerable amount of Förster resonance energy transfer (FRET) was found to occur from the TGA coated MnSe QDs to BSA and with an ensuing efficiency of ∼61%. The origin of anomalous carrier life-time relaxation features has also been encountered through a simplified model as regards head group interaction experienced by the MnSe QDs with different surfactant types. Exploiting luminescence decay characteristics of a magneto-fluorescent candidate could find immense scope in diverse biological applications including assays, labeling and imaging. - Highlights: • Surface anchored manganese selenide quantum dots (MnSe QDs) have been synthesized via a physico-chemical reduction route. • Time resolved luminescence spectra of the QDs have displayed bi-exponential decay trend. • Thioglycolic acid (TGA) coated QDs exhibited shorter lifetime as compared to sodium dodecyl sulfo-succinate (SDS) coated ones. • Upon BSA conjugation, the average life time is four-fold enhanced in MnSe-SDS QDs. • An efficient FRET process has been revealed in BSA conjugated TGA coated MnSe QDs.

Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

Science.gov (United States)

Wong, Louise H; Levine, Tim P

2016-04-15

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

Formation and properties of surface-anchored polymer assemblies with tunable physico-chemical characteristics

Science.gov (United States)

Wu, Tao

We describe two new methodologies leading to the formation of novel surface-anchored polymer assemblies on solid substrates. While the main goal is to understand the fundamentals pertaining to the preparation and properties of the surface-bound polymer assemblies (including neutral and chargeable polymers), several examples also are mentioned throughout the Thesis that point out to practical applications of such structures. The first method is based on generating assemblies comprising anchored polymers with a gradual variation of grafting densities on solid substrates. These structures are prepared by first covering the substrate with a molecular gradient of the polymerization initiator, followed by polymerization from these substrate-bound initiator centers ("grafting from"). We apply this technique to prepare grafting density gradients of poly(acryl amide) (PAAm) and poly(acrylic acid) (PAA) on silica-covered substrates. We show that using the grafting density gradient geometry, the characteristics of surface-anchored polymers in both the low grafting density ("mushroom") regime as well as the high grafting density ("brush") regime can be accessed conveniently on a single sample. We use a battery of experimental methods, including Fourier transform infrared spectroscopy (FTIR), Near-edge absorption fine structure spectroscopy (NEXAFS), contact angle, ellipsometry, to study the characteristics of the surface-bound polymer layers. We also probe the scaling laws of neutral polymer as a function of grafting density, and for weak polyelectrolyte, in addition to the grafting density, we study the affect of solution ionic strength and pH values. In the second novel method, which we coined as "mechanically assisted polymer assembly" (MAPA), we form surface anchored polymers by "grafting from" polymerization initiators deposited on elastic surfaces that have been previously extended uniaxially by a certain length increment, Deltax. Upon releasing the strain in the

Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability

DEFF Research Database (Denmark)

Hansen, Lars; Tawamie, Hasan; Murakami, Yoshiko

2013-01-01

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline...... mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated...... alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous...

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility*

Science.gov (United States)

Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-01-01

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. “Knocking out” PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. PMID:26683373

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

Science.gov (United States)

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-09-15

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Bulk chirality effect for symmetric bistable switching of liquid crystals on topologically self-patterned degenerate anchoring surface.

Science.gov (United States)

Park, Min-Kyu; Joo, Kyung-Il; Kim, Hak-Rin

2017-06-26

We demonstrate a bistable switching liquid crystal (LC) mode utilizing a topologically self-structured dual-groove surface for degenerated easy axes of LC anchoring. In our study, the effect of the bulk elastic distortion of the LC directors on the bistable anchoring surface is theoretically analyzed for balanced bistable states based on a free energy diagram. By adjusting bulk LC chirality, we developed ideally symmetric and stable bistable anchoring and switching properties, which can be driven by a low in-plane pulsed field of about 0.7 V/µm. The fabricated device has a contrast ratio of 196:1.

[Construction of Lactobacillus rhamnosus GG particles surface display system].

Science.gov (United States)

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

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.

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.

Conditional Depletion of Nuclear Proteins by the Anchor Away System (ms# CP-10-0125)

Science.gov (United States)

Fan, Xiaochun; Geisberg, Joseph V.; Wong, Koon Ho; Jin, Yi

2011-01-01

Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. Here we describe a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from nucleus. PMID:21225637

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

Science.gov (United States)

Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-02-19

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Anchoring of alkyl chain molecules on oxide surface using silicon alkoxide

Energy Technology Data Exchange (ETDEWEB)

Narita, Ayumi, E-mail: narita.ayumi@jaea.go.jp [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki-ken 319-1195 (Japan); Graduate School of Science and Engineering, Ibaraki University, Bunnkyo, Mito-shi, Ibaraki-ken 310-8512 (Japan); Baba, Yuji; Sekiguchi, Tetsuhiro; Shimoyama, Iwao; Hirao, Norie [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki-ken 319-1195 (Japan); Yaita, Tsuyoshi [Quantum Beam Science Directorate, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki-ken 319-1195 (Japan); Graduate School of Science and Engineering, Ibaraki University, Bunnkyo, Mito-shi, Ibaraki-ken 310-8512 (Japan)

2012-01-01

Chemical states of the interfaces between octadecyl-triethoxy-silane (ODTS) molecules and sapphire surface were measured by X-ray photoelectron spectroscopy (XPS) and near edge X-ray absorption fine structure (NEXAFS) using synchrotron soft X-rays. The nearly self-assembled monolayer of ODTS was formed on the sapphire surface. For XPS and NEXAFS measurements, it was elucidated that the chemical bond between silicon alkoxide in ODTS and the surface was formed, and the alkane chain of ODTS locates upper side on the surface. As a result, it was elucidated that the silicon alkoxide is a good anchor for the immobilization of organic molecules on oxides.

A cell-surface-anchored ratiometric i-motif sensor for extracellular pH detection.

Science.gov (United States)

Ying, Le; Xie, Nuli; Yang, Yanjing; Yang, Xiaohai; Zhou, Qifeng; Yin, Bincheng; Huang, Jin; Wang, Kemin

2016-06-14

A FRET-based sensor is anchored on the cell surface through streptavidin-biotin interactions. Due to the excellent properties of the pH-sensitive i-motif structure, the sensor can detect extracellular pH with high sensitivity and excellent reversibility.

Different size biomolecules anchoring on porous silicon surface: fluorescence and reflectivity pores infiltration comparative studies

Energy Technology Data Exchange (ETDEWEB)

Giovannozzi, Andrea M.; Rossi, Andrea M. [National Institute for Metrological Research, Thermodynamic Division, Strada delle Cacce 91, 10135 Torino (Italy); Renacco, Chiara; Farano, Alessandro [Ribes Ricecrhe Srl, Via Lavoratori Vittime del Col du Mont 24, 11100 Aosta (Italy); Derosas, Manuela [Biodiversity Srl, Via Corfu 71, 25124 Brescia (Italy); Enrico, Emanuele [National Institute for Metrological Research, Electromagnetism Division, Strada delle Cacce 91, 10135 Torino (Italy)

2011-06-15

The performance of porous silicon optical based biosensors strongly depends on material nanomorphology, on biomolecules distribution inside the pores and on the ability to link sensing species to the pore walls. In this paper we studied the immobilization of biomolecules with different size, such as antibody anti aflatoxin (anti Aflatox Ab, {proportional_to}150 KDa), malate dehydrogenase (MDH, {proportional_to}36KDa) and metallothionein (MT, {proportional_to}6KDa) at different concentrations on mesoporous silicon samples ({proportional_to}15 nm pores diameter). Fluorescence measurements using FITC- labeled biomolecules and refractive index analysis based on reflectivity spectra have been employed together to detect the amount of proteins bound to the surface and to evaluate their diffusion inside the pores. Here we suggest that these two techniques should be used together to have a better understanding of what happens at the porous silicon surface. In fact, when pores dimensions are not perfectly tuned to the protein size a higher fluorescence signal doesn't often correspond to a higher biomolecules distribution inside the pores. When a too much higher concentration of biomolecule is anchored on the surface, steric crowd effects and repulsive interactions probably take over and hinder pores infiltration, inducing a small or absent shift in the fringe pattern even if a higher fluorescence signal is registered. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

Directory of Open Access Journals (Sweden)

Zhen Zhang

Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

CELL-SURFACE DISPLAY OF SYNTHETIC PHYTOCHELATINS USING ICE NUCLEATION PROTEIN FOR ENHANCED HEAVY-METAL BIOACCUMULATION. (R827227)

Science.gov (United States)

Synthetic phytochelatins (ECs) composed of (Glu–Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in i...

Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

Science.gov (United States)

Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

2014-11-01

X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

Science.gov (United States)

Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

2007-06-01

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

Science.gov (United States)

Kitagaki, Hiroshi; Wu, Hong; Shimoi, Hitoshi; Ito, Kiyoshi

2002-11-01

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

DEFF Research Database (Denmark)

Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

2003-01-01

In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protein....... Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resistant, low-density membrane fractions. Our results suggest that O -GlcNAc-modified MSP-1 N-terminal fragments tend to localize...... within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI-linked CD...

A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

DEFF Research Database (Denmark)

Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

2010-01-01

itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology....

Adsorption phenomena and anchoring energy in nematic liquid crystals

CERN Document Server

Barbero, Giovanni

2005-01-01

Despite the large quantity of phenomenological information concerning the bulk properties of nematic phase liquid crystals, little is understood about the origin of the surface energy, particularly the surface, interfacial, and anchoring properties of liquid crystals that affect the performance of liquid crystal devices. Self-contained and unique, Adsorption Phenomena and Anchoring Energy in Nematic Liquid Crystals provides an account of new and established results spanning three decades of research into the problems of anchoring energy and adsorption phenomena in liquid crystals.The book contains a detailed discussion of the origin and possible sources of anchoring energy in nematic liquid crystals, emphasizing the dielectric contribution to the anchoring energy in particular. Beginning with fundamental surface and anchoring properties of liquid crystals and the definition of the nematic phase, the authors explain how selective ion adsorption, dielectric energy density, thickness dependence, and bias voltage...

Position and Orientation Control of a Photo- and Electrochromic Dithienylethene Using a Tripodal Anchor on Gold Surfaces

NARCIS (Netherlands)

Pijper, Thomas C.; Ivashenko, Oleksii; Walko, Martin; Rudolf, Petra; Browne, Wesley R.; Feringa, Ben L.

2015-01-01

A tripodal system for anchoring photochromic dithienylethenes on gold surfaces is reported. The self-assembled monolayers of a tripod-functionalized dithienylethene were characterized by cyclic voltammetry, surface-enhanced Raman spectroscopy (SERS), and X-ray photoelectron spectroscopy (XPS). These

A cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing.

Science.gov (United States)

Ke, Guoliang; Zhu, Zhi; Wang, Wei; Zou, Yuan; Guan, Zhichao; Jia, Shasha; Zhang, Huimin; Wu, Xuemeng; Yang, Chaoyong James

2014-09-10

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid-DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid-DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid-DNA probe showed sensitive and reversible response to pH change in the range of 6.0-8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid-DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid-DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

Microgravity Drill and Anchor System

Science.gov (United States)

Parness, Aaron; Frost, Matthew A.; King, Jonathan P.

2013-01-01

This work is a method to drill into a rock surface regardless of the gravitational field or orientation. The required weight-on-bit (WOB) is supplied by a self-contained anchoring mechanism. The system includes a rotary percussive coring drill, forming a complete sampling instrument usable by robot or human. This method of in situ sample acquisition using micro - spine anchoring technology enables several NASA mission concepts not currently possible with existing technology, including sampling from consolidated rock on asteroids, providing a bolt network for astronauts visiting a near-Earth asteroid, and sampling from the ceilings or vertical walls of lava tubes and cliff faces on Mars. One of the most fundamental parameters of drilling is the WOB; essentially, the load applied to the bit that allows it to cut, creating a reaction force normal to the surface. In every drilling application, there is a minimum WOB that must be maintained for the system to function properly. In microgravity (asteroids and comets), even a small WOB could not be supported conventionally by the weight of the robot or astronaut. An anchoring mechanism would be needed to resist the reactions, or the robot or astronaut would push themselves off the surface and into space. The ability of the system to anchor itself to a surface creates potential applications that reach beyond use in low gravity. The use of these anchoring mechanisms as end effectors on climbing robots has the potential of vastly expanding the scope of what is considered accessible terrain. Further, because the drill is supported by its own anchor rather than by a robotic arm, the workspace is not constrained by the reach of such an arm. Yet, if the drill is on a robotic arm, it has the benefit of not reflecting the forces of drilling back to the arm s joints. Combining the drill with the anchoring feet will create a highly mobile, highly stable, and highly reliable system. The drilling system s anchor uses hundreds of

A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory

NARCIS (Netherlands)

Ostroveanu, Anghelus; Van der Zee, Eddy A.; Dolga, Amalia M.; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Nijholt, Ingrid M.

2007-01-01

A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

Science.gov (United States)

Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

2001-05-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

Science.gov (United States)

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

Science.gov (United States)

Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

2016-06-15

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anchored LH2 complexes in 2D polarization imaging.

Science.gov (United States)

Tubasum, Sumera; Sakai, Shunsuke; Dewa, Takehisa; Sundström, Villy; Scheblykin, Ivan G; Nango, Mamoru; Pullerits, Tõnu

2013-09-26

Protein is a soft material with inherently large structural disorder. Consequently, the bulk spectroscopies of photosynthetic pigment protein complexes provide averaged information where many details are lost. Here we report spectroscopy of single light-harvesting complexes where fluorescence excitation and detection polarizations are both independently rotated. Two samples of peripheral antenna (LH2) complexes from Rhodopseudomonas acidophila were studied. In one, the complexes were embedded in polyvinyl alcohol (PVA) film; in the other, they were anchored on the glass surface and covered by the PVA film. LH2 contains two rings of pigment molecules-B800 and B850. The B800 excitation polarization properties of the two samples were found to be very similar, indicating that orientation statistics of LH2s are the same in these two very different preparations. At the same time, we found a significant difference in B850 emission polarization statistics. We conclude that the B850 band of the anchored sample is substantially more disordered. We argue that both B800 excitation and B850 emission polarization properties can be explained by the tilt of the anchored LH2s due to the spin-casting of the PVA film on top of the complexes and related shear forces. Due to the tilt, the orientation statistics of two samples become similar. Anchoring is expected to orient the LH2s so that B850 is closer to the substrate. Consequently, the tilt-related strain leads to larger deformation and disorder in B850 than in B800.

Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

Science.gov (United States)

Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2015-12-28

Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points.

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

Science.gov (United States)

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2013-08-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

DEFF Research Database (Denmark)

Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

2003-01-01

In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protei...

Surface effects on magnetic Freedericksz transition in ferronematics with soft particle anchoring

International Nuclear Information System (INIS)

Bena, R.-E.; Petrescu, Emil

2003-01-01

The magnetic field-induced Freedericksz transition in a homeotropically aligned ferronematic (FN) in the case of weak anchoring molecules to the walls is studied. A correction to the Burylov-Raikher expression for the second-order transition threshold field is found. The possibility of a first-order transition is discussed in connection with the similar results for nematic liquid crystals. The saturation magnetic field is also obtained in terms of FN, surface and device parameters

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

Science.gov (United States)

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs induce inflammatory responses in avian macrophages.

Directory of Open Access Journals (Sweden)

Yock-Ping Chow

Full Text Available At least 19 glycosylphosphatidylinositol (GPI-anchored surface antigens (SAGs are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown.Ten SAGs, belonging to two previously defined multigene families (A and B, were expressed as soluble recombinant (r fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1β mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity.In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12 may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages.

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

Science.gov (United States)

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a

Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging.

Science.gov (United States)

Gorman, Jason; Fazio, Teresa; Wang, Feng; Wind, Shalom; Greene, Eric C

2010-01-19

Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This "double-tethered" DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein-DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA.

Electro-osmosis of nematic liquid crystals under weak anchoring and second-order surface effects

Science.gov (United States)

Poddar, Antarip; Dhar, Jayabrata; Chakraborty, Suman

2017-07-01

Advent of nematic liquid crystal flows has attracted renewed attention in view of microfluidic transport phenomena. Among various transport processes, electro-osmosis stands as one of the efficient flow actuation mechanisms through narrow confinements. In the present study, we explore the electrically actuated flow of an ordered nematic fluid with ionic inclusions, taking into account the influences from surface-induced elasticity and electrical double layer (EDL) phenomena. Toward this, we devise the coupled flow governing equations from fundamental free-energy analysis, considering the contributions from first- and second-order elastic, dielectric, flexoelectric, charged surface polarization, ionic and entropic energies. The present study focuses on the influence of surface charge and elasticity effects in the resulting linear electro-osmosis through a slit-type microchannel whose surfaces are chemically treated to display a homeotropic-type weak anchoring state. An optical periodic stripe configuration of the nematic director has been observed, especially for higher electric fields, wherein the Ericksen number for the dynamic study is restricted to the order of unity. Contrary to the isotropic electrolytes, the EDL potential in this case was found to be dependent on the external field strength. Through a systematic investigation, we brought out the fact that the wavelength of the oscillating patterns is dictated mainly by the external field, while the amplitude depends on most of the physical variables ranging from the anchoring strength and the flexoelectric coefficients to the surface charge density and electrical double layer thickness.

Synthesis in situ of gold nanoparticles by a dialkynyl Fischer carbene complex anchored to glass surfaces

International Nuclear Information System (INIS)

Bertolino, María Candelaria; Granados, Alejandro Manuel

2016-01-01

Highlights: • Fischer carbene 1-W reacts via cycloaddition without Cu(I) with azide terminal surface. • This reaction on the surface is regioselective to internal triple bond of 1-W. • 1-W bound to glass surface produce AuNps in situ fixed to the surface. • This ability is independent of how 1-W is bonded to the surface. • This hybrid surface can be valuable as SERS substrate or in heterogeneous catalysis. - Abstract: In this work we present a detailed study of classic reactions such as “click reaction” and nucleophilic substitution reaction but on glass solid surface (slides). We used different reactive center of a dialkynylalcoxy Fischer carbene complex of tungsten(0) to be anchored to modified glass surface with amine, to obtain aminocarbene, and azide terminal groups. These cycloaddition reaction showed regioselectivity to internal triple bond of dialkynyl Fischer carbene complex without Cu(I) as catalyst. Anyway the carbene anchored was able to act as a reducing agent to produce in situ very stable gold nanoparticles fixed on surface. We showed the characterization of modified glasses by contact angle measurements and XPS. Synthesized nanoparticles were characterized by SEM, XPS, EDS and UV–vis. The modified glasses showed an important enhancement Raman-SERS. This simple, fast and robust method to create a polifunctional and hybrid surfaces can be valuable in a wide range of applications such as Raman-SERS substrates and other optical fields.

Synthesis in situ of gold nanoparticles by a dialkynyl Fischer carbene complex anchored to glass surfaces

Energy Technology Data Exchange (ETDEWEB)

Bertolino, María Candelaria, E-mail: cbertolino@fcq.unc.edu.ar; Granados, Alejandro Manuel, E-mail: ale@fcq.unc.edu.ar

2016-10-15

Highlights: • Fischer carbene 1-W reacts via cycloaddition without Cu(I) with azide terminal surface. • This reaction on the surface is regioselective to internal triple bond of 1-W. • 1-W bound to glass surface produce AuNps in situ fixed to the surface. • This ability is independent of how 1-W is bonded to the surface. • This hybrid surface can be valuable as SERS substrate or in heterogeneous catalysis. - Abstract: In this work we present a detailed study of classic reactions such as “click reaction” and nucleophilic substitution reaction but on glass solid surface (slides). We used different reactive center of a dialkynylalcoxy Fischer carbene complex of tungsten(0) to be anchored to modified glass surface with amine, to obtain aminocarbene, and azide terminal groups. These cycloaddition reaction showed regioselectivity to internal triple bond of dialkynyl Fischer carbene complex without Cu(I) as catalyst. Anyway the carbene anchored was able to act as a reducing agent to produce in situ very stable gold nanoparticles fixed on surface. We showed the characterization of modified glasses by contact angle measurements and XPS. Synthesized nanoparticles were characterized by SEM, XPS, EDS and UV–vis. The modified glasses showed an important enhancement Raman-SERS. This simple, fast and robust method to create a polifunctional and hybrid surfaces can be valuable in a wide range of applications such as Raman-SERS substrates and other optical fields.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

Science.gov (United States)

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Protein kinase C zeta suppresses low- or high-grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring.

Science.gov (United States)

Deevi, Ravi Kiran; Javadi, Arman; McClements, Jane; Vohhodina, Jekaterina; Savage, Kienan; Loughrey, Maurice Bernard; Evergren, Emma; Campbell, Frederick Charles

2018-04-01

Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi-lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low-grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high-grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high-grade morphology in formalin-fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of

Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

Science.gov (United States)

Martins, Nadini Oliveira; Souza, Renata Torres de; Cordero, Esteban Mauricio; Maldonado, Danielle Cortez; Cortez, Cristian; Marini, Marjorie Mendes; Ferreira, Eden Ramalho; Bayer-Santos, Ethel; Almeida, Igor Correia de; Yoshida, Nobuko; Silveira, José Franco da

2015-11-01

The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions. Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion. We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by

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.

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.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

Science.gov (United States)

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces

DEFF Research Database (Denmark)

Lösche, M.; Piepenstock, M.; Diederich, A.

1993-01-01

The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both...... in subphase water molecules and in the alkyl chains of the lipid surface monolayer, was utilized to determine the interface structure on the molecular length scale. In all cases studied, the protein forms monomolecular layers underneath the interface with thickness values of apprx 40 ANG . A systematic...... dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state...

Surface analysis of PEGylated nano-shields on nanoparticles installed by hydrophobic anchors

DEFF Research Database (Denmark)

Ebbesen, M F; Whitehead, Bradley Joseph; Gonzalez, Borja Ballarin

2013-01-01

and cellular interactions. Methods: Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a hydrophilic PEGylated "nano-shield" inserted at different levels by hydrophobic anchoring using either a phospholipid-PEG conjugate or the copolymer PLGA-block-PEG by an emulsification/diffusion method....... Surface and bulk analysis was performed including X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR) and zeta potential. Cellular uptake was investigated in RAW 264.7 macrophages by flow cytometry. Results: Sub-micron nanoparticles were formed and the combination of (NMR...

Robotic Ankle for Omnidirectional Rock Anchors

Science.gov (United States)

Parness, Aaron; Frost, Matthew; Thatte, Nitish

2013-01-01

Future robotic exploration of near-Earth asteroids and the vertical and inverted rock walls of lava caves and cliff faces on Mars and other planetary bodies would require a method of gripping their rocky surfaces to allow mobility without gravitational assistance. In order to successfully navigate this terrain and drill for samples, the grippers must be able to produce anchoring forces in excess of 100 N. Additionally, the grippers must be able to support the inertial forces of a moving robot, as well gravitational forces for demonstrations on Earth. One possible solution would be to use microspine arrays to anchor to rock surfaces and provide the necessary load-bearing abilities for robotic exploration of asteroids. Microspine arrays comprise dozens of small steel hooks supported on individual suspensions. When these arrays are dragged along a rock surface, the steel hooks engage with asperities and holes on the surface. The suspensions allow for individual hooks to engage with asperities while the remaining hooks continue to drag along the surface. This ensures that the maximum possible number of hooks engage with the surface, thereby increasing the load-bearing abilities of the gripper. Using the microspine array grippers described above as the end-effectors of a robot would allow it to traverse terrain previously unreachable by traditional wheeled robots. Furthermore, microspine-gripping robots that can perch on cliffs or rocky walls could enable a new class of persistent surveillance devices for military applications. In order to interface these microspine grippers with a legged robot, an ankle is needed that can robotically actuate the gripper, as well as allow it to conform to the large-scale irregularities in the rock. The anchor serves three main purposes: deploy and release the anchor, conform to roughness or misalignment with the surface, and cancel out any moments about the anchor that could cause unintentional detachment. The ankle design contains a

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.

Roles of A-Kinase Anchoring Proteins and Phosphodiesterases in the Cardiovascular System

Science.gov (United States)

Ercu, Maria; Klussmann, Enno

2018-01-01

A-kinase anchoring proteins (AKAPs) and cyclic nucleotide phosphodiesterases (PDEs) are essential enzymes in the cyclic adenosine 3′-5′ monophosphate (cAMP) signaling cascade. They establish local cAMP pools by controlling the intensity, duration and compartmentalization of cyclic nucleotide-dependent signaling. Various members of the AKAP and PDE families are expressed in the cardiovascular system and direct important processes maintaining homeostatic functioning of the heart and vasculature, e.g., the endothelial barrier function and excitation-contraction coupling. Dysregulation of AKAP and PDE function is associated with pathophysiological conditions in the cardiovascular system including heart failure, hypertension and atherosclerosis. A number of diseases, including autosomal dominant hypertension with brachydactyly (HTNB) and type I long-QT syndrome (LQT1), result from mutations in genes encoding for distinct members of the two classes of enzymes. This review provides an overview over the AKAPs and PDEs relevant for cAMP compartmentalization in the heart and vasculature and discusses their pathophysiological role as well as highlights the potential benefits of targeting these proteins and their protein-protein interactions for the treatment of cardiovascular diseases. PMID:29461511

Characterization of the C-terminal ER membrane anchor of PTP1B

International Nuclear Information System (INIS)

Anderie, Ines; Schulz, Irene; Schmid, Andreas

2007-01-01

The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure

Stretching of a polymer chain anchored to a surface: the massive field theory approach

International Nuclear Information System (INIS)

Usatenko, Zoryana

2014-01-01

Taking into account the well-known correspondence between the field theoretical φ 4 O(n)-vector model in the limit n → 0 and the behaviour of long-flexible polymer chains, the investigation of stretching of an ideal and a real polymer chain with excluded volume interactions in a good solvent anchored to repulsive and inert surfaces is performed. The calculations of the average stretching force which arises when the free end of a polymer chain moves away from a repulsive or inert surface are performed up to one-loop order of the massive field theory approach in fixed space dimensions d = 3. The analysis of the obtained results indicates that the average stretching force for a real polymer chain anchored to a repulsive surface demonstrates different behaviour for the cases z-tilde ≪1 and z-tilde ≫1, where z-tilde =z ′ /R z . Besides, the results obtained in the framework of the massive field theory approach are in good agreement with previous theoretical results for an ideal polymer chain and results of a density functional theory approach for the region of small applied forces when deformation of a polymer chain in the direction of the applied force is not bigger than the linear extension of a polymer chain in this direction. The better agreement between these two methods is observed in the case where the number of monomers increases and the polymer chain becomes longer. (paper)

Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

Science.gov (United States)

Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

2015-07-13

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

Expression and immunological characterisation of Eimeria tenella glycosylphosphatidylinositol-anchored surface antigen-5

Science.gov (United States)

Ho, Sue-Kim; Nathan, Sheila; Wan, Kiew-Lian

2016-11-01

Eimeria tenella is the most pathogenic of the Eimeria species that infect chickens and causes huge economic losses to the poultry industry. The glycosylphosphatidylinositol-anchored surface antigen-5 (SAG5) found on the surface of the parasite has been shown to activate the chicken's immune system. In this study, recombinant SAG5 was expressed, purified and used to investigate the immune-inducing characteristics of the molecule. Chickens were immunized with purified recombinant SAG5 and sera were subjected to Enzyme-linked Immunosorbant Assay (ELISA). Results indicated that specific antibodies against rSAG5 were produced, with IgG detected at a higher level compared to IgA and IgM. Information on the immunological responses elicited by SAG5 provides essential knowledge that will contribute towards the effort to develop more effective strategies against coccidiosis.

Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor.

Science.gov (United States)

Teo, Adrian Kee Keong; Kulkarni, Rohit N

2012-10-17

Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

Comparative Study on Different Slot Forms of Prestressed Anchor Blocks

Science.gov (United States)

Fan, Rong; Si, Jianhui; Jian, Zheng

2018-03-01

In this paper, two models of prestressed pier, rectangular cavity anchor block and arch hollow anchor block are established. The ABAQUS software was used to calculate the stress of the surface of the neck of the pier and the cavity of the anchor block, through comparative analysis. The results show that compared with the rectangular cavity anchor block, the stress of the pier and the cavity can be effectively reduced when the arch hole is used, and the amount of prestressed anchor can be reduced, so as to obtain obvious economic benefits.

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

International Nuclear Information System (INIS)

Saio, Tomohide; Ogura, Kenji; Yokochi, Masashi; Kobashigawa, Yoshihiro; Inagaki, Fuyuhiko

2009-01-01

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

Science.gov (United States)

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

International Nuclear Information System (INIS)

Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

1990-01-01

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

Anchorage Behaviors of Frictional Tieback Anchors in Silty Sand

Science.gov (United States)

Hsu, Shih-Tsung; Hsiao, Wen-Ta; Chen, Ke-Ting; Hu, Wen-Chi; Wu, Ssu-Yi

2017-06-01

Soil anchors are extensively used in geotechnical applications, most commonly serve as tieback walls in deep excavations. To investigate the anchorage mechanisms of this tieback anchor, a constitutive model that considers both strain hardening and softening and volume dilatancy entitled SHASOVOD model, and FLAC3D software are used to perform 3-D numerical analyses. The results from field anchor tests are compared with those calculated by numerical analyses to enhance the applicability of the numerical method. After the calibration, this research carried out the parameter studies by numerical analyses. The numerical results reveal that whether the yield of soil around an anchor develops to ground surface and/or touches the diaphragm wall depending on the overburden depth H and the embedded depth Z of an anchor, this study suggests the minimum overburden and embedded depths to avoid the yield of soils develop to ground surface and/or touch the diaphragm wall. When the embedded depth, overburden depth or fixed length of an anchor increases, the anchorage capacity also increases. Increasing fixed length should be the optimum method to increase the anchorage capacity for fixed length less than 20m. However, when the fixed length of an anchor exceeds 30 m, the increasing rate of anchorage capacity per fixed length decreases, and progressive yield occurs obviously between the fixed length and surrounding soil.

Spin selection at organic spinterface by anchoring group

Energy Technology Data Exchange (ETDEWEB)

Zhang, Zhao; Qiu, Shuai; Miao, Yuan-yuan; Ren, Jun-feng; Wang, Chuan-kui [School of Physics and Electronics, Shandong Normal University, Jinan 250014 (China); Hu, Gui-chao, E-mail: hgc@sdnu.edu.cn [School of Physics and Electronics, Shandong Normal University, Jinan 250014 (China); Institute of Theoretical Physics, Technische Universität Dresden, 01062 Dresden (Germany)

2017-07-01

Highlights: • The sign of interfacial spin polarization can be selected by using different anchoring groups. • A sp{sup 3}-d or sp-d hybridization may occur and induce spin polarization when the anchoring group changes. • Interfacial spin polarization depends on both the type of the outer orbital of the anchoring atom as well as its energy. - Abstract: Control of organic interfacial spin polarization is crucial in organic spintronics. Based on ab initio theory, here we proposed a spin selection at organic interface via anchoring group by adsorbing an organic molecule onto Ni(111) surface. The results demonstrate that either a positive or negative interfacial spin polarization may be obtained by choosing different anchoring groups. The orbital analysis via the projected density of states shows that the interfacial spin polarization is sensitive to the hybridization of the outer orbital of the anchoring atom as well as its energy relative to the d orbital of the ferromagnetic atom. The work indicates a feasible way to realize spin selection at the organic spinterface by anchoring group.

Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

Directory of Open Access Journals (Sweden)

Sander A. A. Kooijmans

2016-03-01

Full Text Available Background: Extracellular vesicles (EVs are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI anchor signal peptides derived from decay-accelerating factor (DAF. EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results: V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression, but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Directory of Open Access Journals (Sweden)

Wieczorek Andrew S

2012-12-01

Full Text Available Abstract Background The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. Results We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. Conclusions The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study

Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

DEFF Research Database (Denmark)

Oberli, Alexander; Zurbrügg, Laura; Rusch, Sebastian

2016-01-01

is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion...... interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P...

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

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

Science.gov (United States)

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

The Analysis Stability of Anchor Retaining Wall

International Nuclear Information System (INIS)

Benamara, F. Z.; Belabed, L

2011-01-01

The construction of anchored retaining walls reach every day in the field of Civil Engineering especially in public works. Their dimensioning and stability are the axes of research for geotechnical. The rule is to reduce the active forces of the slide and increase the effective normal stress on the rupture surface. So that, we anchored tied-back (constituted by steel cables) in the stable ground located under the failure surface and we apply at the top a traction force. This effort can be distributed over the ground surface by means of small plates or massive reinforced concrete. The study of the stability of anchored retaining wall was also performed by using software GEO4. Many cases can be solved using analytical solutions available in the group GEO4 program, but for our standard model solution studied analytically proved unsatisfactory so we used a numerical analysis based on the method of finite element in this program. The results obtained by numerical study were interpreted to identify the precision numerical predictions. Moreover these methods were useful and economics in the realization of reinforced slopes by tied-buck. (author)

Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation.

Science.gov (United States)

Castillon, Guillaume Alain; Michon, Laetitia; Watanabe, Reika

2013-06-01

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

Release of Glycoprotein (GP1 from the Tegumental Surface of Taenia solium by Phospholipase C from Clostridium perfringens Suggests a Novel Protein-Anchor to Membranes

Directory of Open Access Journals (Sweden)

Abraham Landa

2010-01-01

Full Text Available In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC. Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, αmethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD, suggesting a novel anchor to the membrane for the glycoprotein GP1.

The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface

DEFF Research Database (Denmark)

Behrendt, N; Rønne, E; Danø, K

1995-01-01

PA receptor, uPAR, is a cell-surface protein which plays an important role in the localization and regulation of these processes. In the present article a number of established conclusions concerning the structure and function of uPAR are presented, and in addition various models are discussed which might...... explain additional observations for which the mechanisms involved have not yet been clarified experimentally. uPAR is a highly glycosylated, 3-domain protein, anchored in the plasma membrane by a glycolipid moiety. The domain organization is important for efficient ligand-binding, and the NH2-terminal...

Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines

Directory of Open Access Journals (Sweden)

Yusuke Masuishi

2016-06-01

Full Text Available We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs, which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled “Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment” (Masuishi et al., 2016 [1].

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.

First principles investigations on the electronic structure of anchor groups on ZnO nanowires and surfaces

International Nuclear Information System (INIS)

Dominguez, A.; Lorke, M.; Rosa, A. L.; Frauenheim, Th.; Schoenhalz, A. L.; Dalpian, G. M.; Rocha, A. R.

2014-01-01

We report on density functional theory investigations of the electronic properties of monofunctional ligands adsorbed on ZnO-(1010) surfaces and ZnO nanowires using semi-local and hybrid exchange-correlation functionals. We consider three anchor groups, namely thiol, amino, and carboxyl groups. Our results indicate that neither the carboxyl nor the amino group modify the transport and conductivity properties of ZnO. In contrast, the modification of the ZnO surface and nanostructure with thiol leads to insertion of molecular states in the band gap, thus suggesting that functionalization with this moiety may customize the optical properties of ZnO nanomaterials.

Stability Analysis of Anchored Soil Slope Based on Finite Element Limit Equilibrium Method

Directory of Open Access Journals (Sweden)

Rui Zhang

2016-01-01

Full Text Available Under the condition of the plane strain, finite element limit equilibrium method is used to study some key problems of stability analysis for anchored slope. The definition of safe factor in slices method is generalized into FEM. The “true” stress field in the whole structure can be obtained by elastic-plastic finite element analysis. Then, the optimal search for the most dangerous sliding surface with Hooke-Jeeves optimized searching method is introduced. Three cases of stability analysis of natural slope, anchored slope with seepage, and excavation anchored slope are conducted. The differences in safety factor quantity, shape and location of slip surface, anchoring effect among slices method, finite element strength reduction method (SRM, and finite element limit equilibrium method are comparatively analyzed. The results show that the safety factor given by the FEM is greater and the unfavorable slip surface is deeper than that by the slice method. The finite element limit equilibrium method has high calculation accuracy, and to some extent the slice method underestimates the effect of anchor, and the effect of anchor is overrated in the SRM.

Orented immobilization of farnesylated proteins by the thiol-ene reaction

NARCIS (Netherlands)

Weinrich, Dirk; Lin, Po-Chiao; Jonkheijm, Pascal; Nguyen, Uyen T.T.; Schröder, Hendrik; Niemeyer, Christof M.; Alexandrov, Kirill; Goody, Roger; Waldmann, Herbert

2010-01-01

Anchoring the protein: Proteins were immobilized rapidly under mild conditions by thiol-ene photocoupling between S-farnesyl groups attached to a genetically encodable “CAAX-box” tetrapeptide sequence (A is aliphatic) at the C terminus of the protein and surface-exposed thiols (see scheme). This

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

Directory of Open Access Journals (Sweden)

Jing Li

2018-05-01

Full Text Available GPI-Anchored proteins (GPI-APs can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

Science.gov (United States)

Li, Jing; Liu, Xiuhua; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Cao, Dapeng

2018-05-01

GPI-Anchored proteins (GPI-APs) can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

The Ogden Anchor.

Science.gov (United States)

Knudson, W E; Cerniglia, M W; Carro, A

1998-06-01

Many procedures performed by podiatric surgeons today require the use of a soft-tissue anchoring device. In recent years, many new anchoring devices have become available for use in the foot and ankle. The authors introduce a new soft-tissue anchoring device that has yet to be described in the podiatric literature and present two cases in which the new anchor was used.

Monolayer Assemblies of a De Novo Designed 4-alpha-Helix Bundle Carboprotein and Its Sulfur Anchor Fragment on Au(111) Surfaces Addressed by Voltammetry and In Situ Scanning Tunneling Microscopy

DEFF Research Database (Denmark)

Brask, Jesper; Wackerbarth, Hainer; Jensen, Knud J.

2003-01-01

carboprotein without thiol anchor have been prepared and investigated for comparison. Cyclic and differential pulse voltammetry (DPV) of the proteins show desorption peaks around -750 mV (SCE), whereas the thiol anchor desorption peak is at -685 mV. The peaks are by far the highest for thiol monomeric 4-R...

Optimal suture anchor direction in arthroscopic lateral ankle ligament repair.

Science.gov (United States)

Yoshimura, Ichiro; Hagio, Tomonobu; Noda, Masahiro; Kanazawa, Kazuki; Minokawa, So; Yamamoto, Takuaki

2017-05-26

In this study, the distance between the insertion point of the suture anchors and posterior surface of the fibula during arthroscopic lateral ankle ligament repair was investigated on computed tomography (CT) images. The hypothesis of this study was that there is an optimal insertional direction of the suture anchor to avoid anchor-related complications. One hundred eleven ankles of 98 patients who had undergone three-dimensional CT scans for foot or ankle disorders without deformity of the fibula were assessed (59 males, 52 females; median age 25.5 years; age range 12-78 years). The shortest distance from the insertion point of the suture anchor to the deepest point of the fossa/top of the convex aspect of the fibula was measured on the axial plane, tilting from the longitudinal axis of the fibula at 90°, 75°, 60°, and 45°. The distance from the insertion point of the suture anchor to the posterior surface of the fibula was also measured in a direction parallel to the sagittal plane of the lateral surface of the talus on the axial plane, tilting from the longitudinal axis of the fibula at 90°, 75°, 60°, and 45°. The posterior fossa was observed in all cases on the 90° and 75° images. The distance from the insertion point to the posterior surface of the fibula in the parallel direction was 15.0 ± 3.4 mm at 90°, 17.5 ± 3.2 mm at 75°, 21.7 ± 3.3 mm at 60°, and 25.7 ± 3.6 mm at 45°. The posterior points in the parallel direction were located on the posterior fossa in 36.0% of cases at 90°, in 12.6% at 75°, and in 0.0% at 60° and 45°. The suture anchor should be directed from anterior to posterior at an angle of <45° to the longitudinal axis of the fibula, parallel to the lateral surface of the talus, to avoid passing through the fibula. Cohort study, Level III.

Anchoring submersible ultrasonic receivers in river channels with stable substrate

Science.gov (United States)

Bettoli, Phillip William; Scholten, G.D.; Hubbs, D.

2010-01-01

We developed an anchoring system for submersible ultrasonic receivers (SURs) that we placed on the bottom of the riverine reaches of three main-stem reservoirs in the upper Tennessee River. Each anchor consisted of a steel tube (8.9 x 35.6 cm) welded vertically to a round plate of steel (5.1 x 40.6 cm). All seven SURs and their 57-kg anchors were successfully deployed and retrieved three times over 547 d by a dive team employing surface air-breathing equipment and a davit-equipped boat. All of the anchors and their SURs remained stationary over two consecutive winters on the hard-bottom, thalweg sites where they were deployed. The SUR and its anchor at the most downriver site experienced flows that exceeded 2,100 m(3)/s and mean water column velocities of about 0.9 m/s.

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

International Nuclear Information System (INIS)

Wang, Jimin; Li, Yue; Modis, Yorgo

2014-01-01

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

Energy Technology Data Exchange (ETDEWEB)

Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

2014-04-15

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

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.

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.

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

Science.gov (United States)

Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

2014-07-01

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Stability calculation method of slope reinforced by prestressed anchor in process of excavation.

Science.gov (United States)

Li, Zhong; Wei, Jia; Yang, Jun

2014-01-01

This paper takes the effect of supporting structure and anchor on the slope stability of the excavation process into consideration; the stability calculation model is presented for the slope reinforced by prestressed anchor and grillage beam, and the dynamic search model of the critical slip surface also is put forward. The calculation model of the optimal stability solution of each anchor tension of the whole process is also given out, through which the real-time analysis and checking of slope stability in the process of excavation can be realized. The calculation examples indicate that the slope stability is changed with the dynamic change of the design parameters of anchor and grillage beam. So it is relatively more accurate and reasonable by using dynamic search model to determine the critical slip surface of the slope reinforced by prestressed anchor and grillage beam. Through the relationships of each anchor layout and the slope height of various stages of excavation, and the optimal stability solution of prestressed bolt tension design value in various excavation stages can be obtained. The arrangement of its prestressed anchor force reflects that the layout of the lower part of bolt and the calculation of slope reinforcement is in line with the actual. These indicate that the method is reasonable and practical.

Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

KAUST Repository

Börjesson, Karl

2010-09-28

We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption. © 2010 American Chemical Society.

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

Science.gov (United States)

Pinaud, Fabien; Michalet, Xavier; Iyer, Gopal; Margeat, Emmanuel; Moore, Hsiao-Ping; Weiss, Shimon

2009-06-01

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

Photoactive curcumin-derived dyes with surface anchoring moieties used in ZnO nanoparticle-based dye-sensitized solar cells

International Nuclear Information System (INIS)

Ganesh, T.; Kim, Jong Hoon; Yoon, Seog Joon; Kil, Byung-Ho; Maldar, N.N.; Han, Jin Wook; Han, Sung-Hwan

2010-01-01

Photoactive, eco-friendly and high molar extinction coefficient, curcumin-derived dyes (BCMoxo and BCtCM) have been explored in ZnO nanoparticles (NPs)-based dye-sensitized solar cells (DSSCs). The boron complex curcumin dyes modified with di-carboxylic anchor groups (BCtCM) provided surface attachment with a strong UV-vis region absorption than the dye molecule without anchor groups (BCMoxo). Photoanodes primed with poly-dispersive ZnO NPs (∼80-50 nm) specifically devised for these dyes and optimized for the critical thickness, sensitization time and concentration using a solvent-free ionic electrolyte so as to get current density as high as 1.66 mA/cm 2 under 80 mW/cm 2 irradiation. Therefore, a successful conversion of visible light into electricity by using these curcumin-derived dyes (natural derived photoactive molecules) as photosensitizer in DSSCs would be a great interest in future studies for enhancing further conversion efficiencies.

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer.

Science.gov (United States)

Stimac, Adela; Segota, Suzana; Dutour Sikirić, Maja; Ribić, Rosana; Frkanec, Leo; Svetličić, Vesna; Tomić, Srđanka; Vranešić, Branka; Frkanec, Ruža

2012-09-01

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface. Copyright © 2012 Elsevier B.V. All rights reserved.

Anchored PKA as a gatekeeper for gap junctions.

Science.gov (United States)

Pidoux, Guillaume; Taskén, Kjetil

2015-01-01

Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types.

Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

Science.gov (United States)

Wang, Jimin; Li, Yue; Modis, Yorgo

2014-01-01

The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

Influence of weak anchoring upon the alignment of smectic A liquid crystals with surface pretilt

International Nuclear Information System (INIS)

De Vita, R; Stewart, I W

2008-01-01

Equilibrium configurations for smectic A liquid crystals in a 'bookshelf' geometry are determined from a nonlinear continuum model under strong and weak anchoring conditions at the boundary for the usual director n. Natural boundary conditions are derived for n and the smectic layer normal a when a preferred director orientation n p , which generally induces a director pretilt, is prescribed on the boundaries. Two key aspects are examined via the nonlinear equilibrium equations: the separation of n from a and the influence of weak anchoring. The orientations of n and a relative to n p may differ significantly and depend very much upon the magnitude of the anchoring strength. These results from a nonlinear theory are natural and novel developments of previous classical linearized models for which n ≡ a. Comparisons are also drawn between solutions for strong and weak anchoring conditions

Ultraweak azimuthal anchoring of a nematic liquid crystal on a planar orienting photopolymer

International Nuclear Information System (INIS)

Nespoulous, Mathieu; Blanc, Christophe; Nobili, Maurizio

2007-01-01

The search of weak anchoring is an important issue for a whole class of liquid crystal displays. In this paper we present an orienting layer showing unreached weak planar azimuthal anchoring for 4-n-pentyl-4 ' -cyanobiphenyl nematic liquid crystal (5CB). Azimuthal extrapolation lengths as large as 80 μm are easily obtained. Our layers are made with the commercial photocurable polymer Norland optical adhesive 60. The anisotropy of the film is induced by the adsorption of oriented liquid crystal molecules under a 2 T magnetic field applied parallel to the surfaces. We use the width of surface π-walls and a high-field electro-optical method to measure, respectively, the azimuthal and the zenithal anchorings. The azimuthal anchoring is extremely sensitive to the ultraviolet (UV) dose and it also depends on the magnetic field application duration. On the opposite, the zenithal anchoring is only slightly sensitive to the preparation parameters. All these results are discussed in terms of the adsorption/desorption mechanisms of the liquid crystal molecules on the polymer layer and of the flexibility of the polymer network

Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

Directory of Open Access Journals (Sweden)

Preetinder K Dhanoa

Full Text Available BACKGROUND: Tail-anchored (TA proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34 and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9. Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

Science.gov (United States)

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

2013-03-01

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

Anchor Bolt Position in Base Plate In Terms Of T and J Anchor Bolt

Directory of Open Access Journals (Sweden)

b Osman Mohamad Hairi

2017-01-01

Full Text Available Generally, L anchor bolt system has been used for a long period of time in construction industry as one of the distributing load structures. However, there are some weaknesses in L anchor bolt which may straighten and pullup when charged with tensile load. Current practices prefer to use other types of anchor bolt systems, such as headed studs anchor bolt system to replace the L anchor bolt design. There has been lack of studies to prove that it is more effective in terms of performance. A new T anchor bolt which was basically modified from headed studs anchor bolt was proposed in this study to compare its performance of tensile loading in concrete failure to typical L design. This study aims to determine whether the T anchor bolt system gives better performance as compared to an L anchor bolt system. The performance was rated based on tensile loading on concrete failure pattern. A pullout test was conducted on two different anchor bolt systems, namely L and T. The anchor bolt embedded depth, h in concrete were varied according to their hook or bend radius. Each sample was repeated twice. There were totally eight samples. The hook or bend radius used were 50 mm and 57.5 mm for sample L1 and L2, respectively. 90-degree bend were used on sample T1 and T2. Based on test results, it can be seen that the performance of concrete failure pattern under tensile load on both L and T anchor bolt design samples with 200 mm embedment depth was better than deeper embedment depth of 230 mm. But the L anchor bolt design gives the best results as compared to T design. Although T anchor bolt design shows higher resistance before first bond failure to the concrete sample. T anchor bolt was analysed and needed deeper embedment depth to allow formation of cone pull-out shape to acquire better performance.

Failure Capacity Evaluation for Anchor System of NPP Facilities by using a Shaking Table Test

International Nuclear Information System (INIS)

Kwon, Hyung O; Jung, Min Ki; Park, Jin Wan; Lim, Ji Hoon

2010-02-01

This study investigate the destructive influence of crack locations on the anchor performance to evaluate the seismic performance of NPP equipment anchored on damaged concrete. For this purpose, small-scale specimens were fabricated according to the following three cases: 1) with a non-damaged anchor; 2) with cracks running through the anchor; and 3) with cracks along the expected corn-shape fracture away from the anchor. The result verified with the finite element method is as follows: In the first and second cases that is, with a non-damaged anchor and with cracks running through the anchor destruction occurred at the anchor steel. In the third case that is, with cracks around the anchor, a 30% decline in the seismic performance was identified. This result indicates that evaluation of seismic performance and relevant reinforcement are required when cracks occur away from the anchor along the expected destructive surface

Root hairs aid soil penetration by anchoring the root surface to pore walls.

Science.gov (United States)

Bengough, A Glyn; Loades, Kenneth; McKenzie, Blair M

2016-02-01

The physical role of root hairs in anchoring the root tip during soil penetration was examined. Experiments using a hairless maize mutant (Zea mays: rth3-3) and its wild-type counterpart measured the anchorage force between the primary root of maize and the soil to determine whether root hairs enabled seedling roots in artificial biopores to penetrate sandy loam soil (dry bulk density 1.0-1.5g cm(-3)). Time-lapse imaging was used to analyse root and seedling displacements in soil adjacent to a transparent Perspex interface. Peak anchorage forces were up to five times greater (2.5N cf. 0.5N) for wild-type roots than for hairless mutants in 1.2g cm(-3) soil. Root hair anchorage enabled better soil penetration for 1.0 or 1.2g cm(-3) soil, but there was no significant advantage of root hairs in the densest soil (1.5g cm(-3)). The anchorage force was insufficient to allow root penetration of the denser soil, probably because of less root hair penetration into pore walls and, consequently, poorer adhesion between the root hairs and the pore walls. Hairless seedlings took 33h to anchor themselves compared with 16h for wild-type roots in 1.2g cm(-3) soil. Caryopses were often pushed several millimetres out of the soil before the roots became anchored and hairless roots often never became anchored securely.The physical role of root hairs in anchoring the root tip may be important in loose seed beds above more compact soil layers and may also assist root tips to emerge from biopores and penetrate the bulk soil. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Decay accelerating factor (DAF) is anchored to membranes by a C-terminal glycolipid

International Nuclear Information System (INIS)

Medof, M.E.; Haas, R.; Walter, E.I.; Rosenberry, T.L.

1986-01-01

Purified 70 kDa membrane (m) DAF incorporates into cells when added in vitro. A 2 kDa smaller DAF form which functions extrinsically like C4bp but is unable to incorporate can be isolated from urine (u). Because of common deficits of mDAF and acetylcholinesterase (AChE) in erythrocytes (E) of patients with paroxysmal nocturnal hemoglobinuria (PNH), mDAF was analyzed for a O-terminal glycolipid membrane anchor similar to that in E AChE. Incubation of E with phosphatidylinositol-specific phospholipase C, an enzyme which cleaves a similar glycolipid anchor in trypanosome variant surface glycoproteins (mfVSGs), released 20% of the DAF antigen. The released DAF species resembled uDAF in size, extrinsic model of C4b2a decay, and lack of hydrophobicity. Reductive radiomethylation of mDAF with [ 14 C]HCHO and NaCNBH 3 revealed ethanolamine and glucosamine in proportions similar to those in the E AChE glycolipid anchor. Papain cleavage of radiomethylated mDAF released the labeled ethanolamine and glucosamine in small O-terminal fragments from the residual DAF that retained N-terminal Asp. Following labeling of the anchors of mDAF and E AChE with the lipophilic photoreagent 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl)diazirine, cleavage at the glucosamine residue by deamination quantitatively released the label from both proteins. Biosynthetic labeling of Hela cells with [ 3 H]ethanolamine resulted in rapid 3 H incorporation into both 48 kDa proDAF and 70 kDa mDAF. These data indicate that mDAF is anchored by a glycolipid similar to that in E AChE, mfVSGs and Thy-1 antigen and raise the possibility that a defect in the assembly or attachment of this structure could account for the deficits of mDAF and E AChE in PNH

Influence of weak anchoring upon the alignment of smectic A liquid crystals with surface pretilt

Energy Technology Data Exchange (ETDEWEB)

De Vita, R [Department of Engineering Science and Mechanics, Virginia Tech, 230 Norris Hall, Blacksburg, VA 24061 (United States); Stewart, I W [Department of Mathematics, University of Strathclyde, Livingstone Tower, 26 Richmond Street, Glasgow G1 1XH (United Kingdom)], E-mail: devita@vt.edu, E-mail: i.w.stewart@strath.ac.uk

2008-08-20

Equilibrium configurations for smectic A liquid crystals in a 'bookshelf' geometry are determined from a nonlinear continuum model under strong and weak anchoring conditions at the boundary for the usual director n. Natural boundary conditions are derived for n and the smectic layer normal a when a preferred director orientation n{sub p}, which generally induces a director pretilt, is prescribed on the boundaries. Two key aspects are examined via the nonlinear equilibrium equations: the separation of n from a and the influence of weak anchoring. The orientations of n and a relative to n{sub p} may differ significantly and depend very much upon the magnitude of the anchoring strength. These results from a nonlinear theory are natural and novel developments of previous classical linearized models for which n {identical_to} a. Comparisons are also drawn between solutions for strong and weak anchoring conditions.

A Proteomics Investigation of Anchored PKA-RI Signaling

NARCIS (Netherlands)

Kovanich, D.

2013-01-01

Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring

Photoactive curcumin-derived dyes with surface anchoring moieties used in ZnO nanoparticle-based dye-sensitized solar cells

Energy Technology Data Exchange (ETDEWEB)

Ganesh, T.; Kim, Jong Hoon; Yoon, Seog Joon; Kil, Byung-Ho; Maldar, N.N. [Inorganic Nano-Materials Laboratory, Department of Chemistry, Hanyang University, Sung-Dong-Ku, Haengdang-dong 17, Seoul (Korea, Republic of); Han, Jin Wook, E-mail: jwhan@hanyang.ac.kr [Inorganic Nano-Materials Laboratory, Department of Chemistry, Hanyang University, Sung-Dong-Ku, Haengdang-dong 17, Seoul (Korea, Republic of); Han, Sung-Hwan, E-mail: shhan@hanyang.ac.kr [Inorganic Nano-Materials Laboratory, Department of Chemistry, Hanyang University, Sung-Dong-Ku, Haengdang-dong 17, Seoul (Korea, Republic of)

2010-09-01

Photoactive, eco-friendly and high molar extinction coefficient, curcumin-derived dyes (BCMoxo and BCtCM) have been explored in ZnO nanoparticles (NPs)-based dye-sensitized solar cells (DSSCs). The boron complex curcumin dyes modified with di-carboxylic anchor groups (BCtCM) provided surface attachment with a strong UV-vis region absorption than the dye molecule without anchor groups (BCMoxo). Photoanodes primed with poly-dispersive ZnO NPs ({approx}80-50 nm) specifically devised for these dyes and optimized for the critical thickness, sensitization time and concentration using a solvent-free ionic electrolyte so as to get current density as high as 1.66 mA/cm{sup 2} under 80 mW/cm{sup 2} irradiation. Therefore, a successful conversion of visible light into electricity by using these curcumin-derived dyes (natural derived photoactive molecules) as photosensitizer in DSSCs would be a great interest in future studies for enhancing further conversion efficiencies.

Anchored but not internalized: shape dependent endocytosis of nanodiamond

Science.gov (United States)

Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

2017-04-01

Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

Protein adsorption resistant surface on polymer composite based on 2D- and 3D-controlled grafting of phospholipid moieties

International Nuclear Information System (INIS)

Hoshi, Toru; Matsuno, Ryosuke; Sawaguchi, Takashi; Konno, Tomohiro; Takai, Madoka; Ishihara, Kazuhiko

2008-01-01

To prepare the biocompatible surface, a phosphorylcholine (PC) group was introduced on this hydroxyl group generated by surface hydrolysis on the polymer composite composed of polyethylene (PE) and poly (vinyl acetate) (PVAc) prepared by supercritical carbon dioxide. Two different procedures such as two-dimensional (2D) modification and three-dimensional (3D) modification were applied to obtain the steady biocompatible surface. 2D modification was that PC groups were directly anchored on the surface of the polymer composite. 3D modification was that phospholipid polymer was grafted from the surface of the polymer composite by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-methacryloyloxyethyl phosphorylcholine (MPC). The surfaces were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle measurements, and atomic force microscope. The effects of the poly(MPC) chain length on the protein adsorption resistivity were investigated. The protein adsorption on the polymer composite surface with PC groups modified by 2D or 3D modification was significantly reduced as compared with that on the unmodified PE. Further, the amount of protein adsorbed on the 3D modified surface that is poly(MPC)-grafted surface decreased with an increase in the chain length of the poly(MPC). The surface with an arbitrary structure and the characteristic can be constructed by using 2D and 3D modification. We conclude that the polymer composites of PE/PVAc with PC groups on the surface are useful for fabricating biomedical devices due to their good mechanical and surface properties

ANTS-anchored Zn-Al-CO3-LDH particles as fluorescent probe for sensing of folic acid

International Nuclear Information System (INIS)

Liu, Pengfei; Liu, Dan; Liu, Yanhuan; Li, Lei

2016-01-01

A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO 3 -layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn 2+ ions of Zn-Al-CO 3 -LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO 3 -LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO 3 -LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO 3 groups in ANTS-anchored on the surface of Zn-Al-CO 3 -LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO 3 -LDH particles can afford a very sensitive system for the sensing FA in aqueous solution. - Highlights: • A novel fluorescent nanosensor has been developed. • The sensor exhibited highly sensitive and selective response to FA. • The fluorescence quenching was fitted to Stern–Volmer equation. • The linear response range was 1–200 μM with a limit of detection of 0.1 μM.

Density control of dodecamanganese clusters anchored on silicon(100).

Science.gov (United States)

Condorelli, Guglielmo G; Motta, Alessandro; Favazza, Maria; Nativo, Paola; Fragalà, Ignazio L; Gatteschi, Dante

2006-04-24

A synthetic strategy to control the density of Mn12 clusters anchored on silicon(100) was investigated. Diluted monolayers suitable for Mn12 anchoring were prepared by Si-grafting mixtures of the methyl 10-undecylenoate precursor ligand with 1-decene spectator spacers. Different ratios of these mixtures were tested. The grafted surfaces were hydrolyzed to reveal the carboxylic groups available for the subsequent exchange with the [Mn12O12(OAc)16(H2O)4]4 H2O2 AcOH cluster. Modified surfaces were analyzed by attenuated total reflection (ATR)-FTIR spectroscopy, X-ray photoemission spectroscopy (XPS), and AFM imaging. Results of XPS and ATR-FTIR spectroscopy show that the surface mole ratio between grafted ester and decene is higher than in the source solution. The surface density of the Mn12 cluster is, in turn, strictly proportional to the ester mole fraction. Well-resolved and isolated clusters were observed by AFM, using a diluted ester/decene 1:1 solution.

Numerical Simulation of Electro-Mechanical Impedance Response in Cable-Anchor Connection Interface

International Nuclear Information System (INIS)

Nguyen, Khac Duy; Kim, Jeong Tae

2011-01-01

In this study, a finite element(FE) analysis on electro-mechanical impedance response of cable-anchor connection interface under various anchor force is presented. In order to achieve the objective, the following approaches are implemented. Firstly, an interface washer coupled with piezoelectric(PZT) material is designed for monitoring cable-force loss. The interface washer is a small aluminum plate on which a PZT patch is surface-bonded. Cable-force loss could be monitored by installing the interface washer between the anchor plate and the anchorage of cable-anchor connection and examining the changes of impedance of the interface washer. Secondly, a FE model for cable-anchor connection is established to examine the effect of cable-force on impedance response of interface washer. Also, the effects of geometrical and material properties of the interface washer on impedance responses under various cable-forces are investigated. Finally, validation of the FE analysis is experimentally evaluated by a lab-scale cable-anchor connection

Monolayers of a De Novo Designed 4-Alpha-Helix Bundle Carboprotein and Partial Structures on Au(111)-Surfaces

DEFF Research Database (Denmark)

Brask, Jesper; Wackerbarth, Hainer; Jensen, Knud Jørgen

2002-01-01

on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The galactopyranoside with thiol anchor and the thiol anchor alone were prepared for comparison. Voltammetry of the three molecules on Au(111) showed reductive desorption peaks caused by monolayer adsorption via...... thiolate-Au bonding. In situ STM of the thiol anchor disclosed an ordered adlayer with clear domains and molecular features. This holds promise, broadly for single-molecule voltammetry and the SPM and scanning tunnelling microscopy (STM) of natural and synthetic proteins....

Analysis of Anchoring Mechanism of Fully Grouted Prestressed Anchor

Directory of Open Access Journals (Sweden)

WEN Zhi-jie

2014-01-01

Full Text Available Some researchers have been carried out on analysis of the influence of the full grouted prestressed anchor shape of borehole wall on its carrying capacity. Based on the self-affine fractal feature of anchor borehole wall structural plane, the relation equation among structural plane shear strength, liquid injection pressure, tensile load and structural plane fractal dimension D was built, the instability judgment criterion of anchoring bearing strata and rock structural plane was determined, the solving equations of disintegrated rock support density were derived. Based on the experimental results, the theoretical basis of support design under the disintegrated rock condition was offered.

Modeling and experimental validation of sawing based lander anchoring and sampling methods for asteroid exploration

Science.gov (United States)

Zhang, Jun; Dong, Chengcheng; Zhang, Hui; Li, Song; Song, Aiguo

2018-05-01

This paper presents a novel lander anchoring system based on sawing method for asteroid exploration. The system is composed of three robotic arms, three cutting discs, and a control system. The discs mounted at the end of the arms are able to penetrate into the rock surface of asteroids. After the discs cut into the rock surface, the self-locking function of the arms provides forces to fix the lander on the surface. Modeling, trajectory planning, simulations, mechanism design, and prototype fabrication of the anchoring system are discussed, respectively. The performances of the system are tested on different kinds of rocks, at different sawing angles, locations, and speeds. Results show that the system can cut 15 mm deep into granite rock in 180 s at sawing angle of 60°, with the average power of 58.41 W, and the "weight on bit" (WOB) of 8.637 N. The 7.8 kg anchoring system is capable of providing omni-directional anchoring forces, at least 225 N normal and 157 N tangent to the surface of the rock. The system has the advantages of low-weight, low energy consumption and balance forces, high anchoring efficiency and reliability, and could enable the lander to move and sample or assist astronauts and robots in walking and sampling on asteroids.

Anchors as Semantic Primes in Value Construction: An EEG Study of the Anchoring Effect.

Directory of Open Access Journals (Sweden)

Qingguo Ma

Full Text Available Previous research regarding anchoring effects has demonstrated that human judgments are often assimilated to irrelevant information. Studies have demonstrated that anchors influence the economic valuation of various products and experiences; however, the cognitive explanations of this effect remain controversial, and its neural mechanisms have rarely been explored. In the current study, we conducted an electroencephalography (EEG experiment to investigate the anchoring effect on willingness to accept (WTA for an aversive hedonic experience and the role of anchors in this judgment heuristic. The behavioral results demonstrated that random numbers affect participants' WTA for listening to pieces of noise. The participants asked for higher pay after comparing their WTA with higher numbers. The EEG results indicated that anchors also influenced the neural underpinnings of the valuation process. Specifically, when a higher anchor number was drawn, larger P2 and late positive potential amplitudes were elicited, reflecting the anticipation of more intensive pain from the subsequent noise. Moreover, higher anchors induced a stronger theta band power increase compared with lower anchors when subjects listened to the noises, indicating that the participants felt more unpleasant during the actual experience of the noise. The levels of unpleasantness during both anticipation and experience were consistent with the semantic information implied by the anchors. Therefore, these data suggest that a semantic priming process underlies the anchoring effect in WTA. This study provides proof for the robustness of the anchoring effect and neural evidence of the semantic priming model. Our findings indicate that activated contextual information, even seemingly irrelevant, can be embedded in the construction of economic value in the brain.

Susceptibility to anchoring effects

Directory of Open Access Journals (Sweden)

Todd McElroy

2007-02-01

Full Text Available Previous research on anchoring has shown this heuristic to be a very robust psychological phenomenon ubiquitous across many domains of human judgment and decision-making. Despite the prevalence of anchoring effects, researchers have only recently begun to investigate the underlying factors responsible for how and in what ways a person is susceptible to them. This paper examines how one such factor, the Big-Five personality trait of openness-to-experience, influences the effect of previously presented anchors on participants' judgments. Our findings indicate that participants high in openness-to-experience were significantly more influenced by anchoring cues relative to participants low in this trait. These findings were consistent across two different types of anchoring tasks providing convergent evidence for our hypothesis.

Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

Science.gov (United States)

Aberle, Daniel; Oetter, Kay-Marcus; Meyers, Gregor

2015-01-01

Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

Directory of Open Access Journals (Sweden)

Daniel Aberle

Full Text Available Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

Comparison of Suture-Based Anchors and Traditional Bioabsorbable Anchors in Foot and Ankle Surgery.

Science.gov (United States)

Hembree, W Chad; Tsai, Michael A; Parks, Brent G; Miller, Stuart D

We compared the pullout strength of a suture-based anchor versus a bioabsorbable anchor in the distal fibula and calcaneus and evaluated the relationship between bone mineral density and peak load to failure. Eight paired cadaveric specimens underwent a modified Broström procedure and Achilles tendon reattachment. The fibula and calcaneus in the paired specimens received either a suture-based anchor or a bioabsorbable suture anchor. The fibular and calcaneal specimens were loaded to failure, defined as a substantial decrease in the applied load or pullout from the bone. In the fibula, the peak load to failure was significantly greater with the suture-based versus the bioabsorbable anchors (133.3 ± 41.8 N versus 76.8 ± 35.3 N; p = .002). No significant difference in load with 5 mm of displacement was found between the 2 groups. In the calcaneus, no difference in the peak load to failure was found between the 2 groups, and the peak load to failure with 5 mm of displacement was significantly lower with the suture-based than with the bioabsorbable anchors (52.2 ± 9.8 N versus 75.9 ± 12.4 N; p = .003). Bone mineral density and peak load to failure were significantly correlated in the fibula with the suture-based anchor. An innovative suture-based anchor had a greater peak load to failure compared with a bioabsorbable anchor in the fibula. In the calcaneus, the load at 5 mm of displacement was significantly lower in the suture-based than in the bioabsorbable group. The correlation findings might indicate the need for a cortical bone shelf with the suture-based anchor. Suture-based anchors could be a viable alternative to bioabsorbable anchors for certain foot and ankle procedures. Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

ANTS-anchored Zn-Al-CO{sub 3}-LDH particles as fluorescent probe for sensing of folic acid

Energy Technology Data Exchange (ETDEWEB)

Liu, Pengfei; Liu, Dan; Liu, Yanhuan [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China); Li, Lei, E-mail: lilei@mail.buct.edu.cn [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029 (China)

2016-09-15

A novel fluorescent nanosensor for detecting folic acid (FA) in aqueous media has been developed based on 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) anchored to the surface of Zn-Al-CO{sub 3}-layered double hydroxides (LDH) particles. The nanosensor showed high fluorescence intensity and good photostability due to a strong coordination interaction between surface Zn{sup 2+} ions of Zn-Al-CO{sub 3}-LDH and N atoms of ANTS, which were verified by result of X-ray photoelectron spectroscopy (XPS). ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH restricted the intra-molecular rotation leading to ANTS-anchored J-type aggregation emission enhancement. ANTS-anchored Zn-Al-CO{sub 3}-LDH particles exhibited highly sensitive and selective response to FA over other common metal ions and saccharides present in biological fluids. The proposed mechanism was that oxygen atoms of -SO{sub 3} groups in ANTS-anchored on the surface of Zn-Al-CO{sub 3}-LDH were easily collided by FA molecules to form potential hydrogen bonds between ANTS-anchored and FA molecules, which could effectively quench the ANTS-anchored fluorescence. Under the simulated physiological conditions (pH of 7.4), the fluorescence quenching was fitted to Stern-Volmer equation with a linear response in the concentration range of 1 μM to 200 μM with a limit of detection of 0.1 μM. The results indicate that ANTS-anchored Zn-Al-CO{sub 3}-LDH particles can afford a very sensitive system for the sensing FA in aqueous solution. - Highlights: • A novel fluorescent nanosensor has been developed. • The sensor exhibited highly sensitive and selective response to FA. • The fluorescence quenching was fitted to Stern–Volmer equation. • The linear response range was 1–200 μM with a limit of detection of 0.1 μM.

Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment

DEFF Research Database (Denmark)

Larsen, Jannik B.; Kennard, Celeste; Pedersen, Søren L.

2017-01-01

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We...

Molecular evolution of a-kinase anchoring protein (AKAP-7: implications in comparative PKA compartmentalization

Directory of Open Access Journals (Sweden)

Johnson Keven R

2012-07-01

Full Text Available Abstract Background A-Kinase Anchoring Proteins (AKAPs are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA, directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18 are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. Results Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels that promote the production of AKAP7γ instead of AKAP7δ. Conclusions This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

Science.gov (United States)

Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

2014-01-01

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

Reinforcing mechanism of anchors in slopes: a numerical comparison of results of LEM and FEM

Science.gov (United States)

Cai, Fei; Ugai, Keizo

2003-06-01

This paper reports the limitation of the conventional Bishop's simplified method to calculate the safety factor of slopes stabilized with anchors, and proposes a new approach to considering the reinforcing effect of anchors on the safety factor. The reinforcing effect of anchors can be explained using an additional shearing resistance on the slip surface. A three-dimensional shear strength reduction finite element method (SSRFEM), where soil-anchor interactions were simulated by three-dimensional zero-thickness elasto-plastic interface elements, was used to calculate the safety factor of slopes stabilized with anchors to verify the reinforcing mechanism of anchors. The results of SSRFEM were compared with those of the conventional and proposed approaches for Bishop's simplified method for various orientations, positions, and spacings of anchors, and shear strengths of soil-grouted body interfaces. For the safety factor, the proposed approach compared better with SSRFEM than the conventional approach. The additional shearing resistance can explain the influence of the orientation, position, and spacing of anchors, and the shear strength of soil-grouted body interfaces on the safety factor of slopes stabilized with anchors.

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

Science.gov (United States)

Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-01-01

ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

Directory of Open Access Journals (Sweden)

Pegine Walrad

2009-02-01

Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

Composite materials formed with anchored nanostructures

Science.gov (United States)

Seals, Roland D; Menchhofer, Paul A; Howe, Jane Y; Wang, Wei

2015-03-10

A method of forming nano-structure composite materials that have a binder material and a nanostructure fiber material is described. A precursor material may be formed using a mixture of at least one metal powder and anchored nanostructure materials. The metal powder mixture may be (a) Ni powder and (b) NiAl powder. The anchored nanostructure materials may comprise (i) NiAl powder as a support material and (ii) carbon nanotubes attached to nanoparticles adjacent to a surface of the support material. The process of forming nano-structure composite materials typically involves sintering the mixture under vacuum in a die. When Ni and NiAl are used in the metal powder mixture Ni.sub.3Al may form as the binder material after sintering. The mixture is sintered until it consolidates to form the nano-structure composite material.

Experimental Research on Destruction Mode and Anchoring Performance of Carbon Fiber Phyllostachys pubescens Anchor Rod with Different Forms

Directory of Open Access Journals (Sweden)

Wang Yulan

2018-01-01

Full Text Available The anchoring technology is extensively applied in reinforcing protection of the earth relics. Now that no specification is available for different new anchor rods in earth relics protection due to diversified destruction modes of earth relics and complexity of engineering technology conditions, it is urgent to guide reinforcing design and construction with a complete detailed anchor rod research document. With the new carbon fiber Phyllostachys pubescens anchor rod as the research object, six lots of in situ tests are designed to, respectively, study the destruction mode and anchoring performance of the carbon fiber Phyllostachys pubescens anchor rod under different anchor length L, anchor rod diameter D, bore diameter H, grouting material S, rib spacing R, and inclination angle A in this paper. By studying load shift curve experiment in drawing of the anchor rod, the destruction mode and ultimate bearing capacity of the carbon fiber Phyllostachys pubescens anchor rod in different experiment lots are obtained, and the concept of permitted application value N in anchor rod design is proposed. By studying strain distribution characteristics of anchor rods in experimental lots along the length direction under action of the permitted application value N and combining the existing destruction mode and ultimate bearing capacity, this paper analyzes influences of L, D, H, S, R, and A on anchoring effect of the carbon fiber Phyllostachys pubescens anchor rod; gives the reasonable value range of L, D, H, and R when the carbon fiber Phyllostachys pubescens anchor rod is used for reinforcing design of the earth relics; and provides favorable experiment basis for reinforcing design of the earth relics based on the carbon fiber Phyllostachys pubescens anchor rod.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

Directory of Open Access Journals (Sweden)

Chisato Morita

Full Text Available Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease, and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs. Recombinant SWAN protein (rSWAN digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+ and Ca(2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

Science.gov (United States)

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Yeast cell surface display for lipase whole cell catalyst and its applications

Energy Technology Data Exchange (ETDEWEB)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

2014-08-01

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

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

Experimental testing of anchoring devices for bottom rails in partially anchored timber frame shear walls

OpenAIRE

Caprolu, Giuseppe

2011-01-01

Källsner and Girhammar have presented a new plastic design method of wood-framed shear walls at ultimate limit state. This method allows the designer to calculate the load-carrying capacity of shear walls partially anchored, where the leading stud is not anchored against the uplift.The anchorage system of shear walls is provided from anchor bolts and hold downs. Anchor bolts provide horizontal shear continuity between the bottom rail and the foundation. Hold downs are directly connected from ...

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...

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

DEFF Research Database (Denmark)

Willumsen, B M; Papageorge, A G; Hubbert, N

1985-01-01

or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

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.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

Directory of Open Access Journals (Sweden)

Markus M. Knodel

2018-01-01

Full Text Available Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Science.gov (United States)

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

Science.gov (United States)

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

KAUST Repository

Knodel, Markus

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

Science.gov (United States)

Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

2008-01-04

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

Traction endurance biomechanical study of metallic suture anchors at different insertion angles

Directory of Open Access Journals (Sweden)

Azato Flávia Namie

2003-01-01

Full Text Available The suture anchors' insertion angle and its traction resistance are the main subjects of this study. Twenty trials were realized using threaded suture anchors in four diferents angulations (30º /45º /60º /90º in human bone (distal femur and another twenty trials in artificial bone (SawboneTM. The anchors were pulled out being tractioned uprightly from its bone surface by a Kratos Universal test machine. The human bone results found no relation between the main subjects of this study, so whithout statistical value. On the other hand at the artificial bone the insertion angle of 90º beared more traction, being statistically significant compared to the other angles.

Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

Directory of Open Access Journals (Sweden)

Muscariello Lidia

2006-05-01

Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris

Science.gov (United States)

To construct the Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE), the gene for the anchor protein (AGa1) was obtained from Saccharomyces cerevisiae and was fused with the modified Bombyx mori acetylcholinesterase gene (bmace) and transformed int...

Side-by-side comparison of Raman spectra of anchored and suspended carbon nanomaterials

International Nuclear Information System (INIS)

Sidorov, Anton N; Pabba, Santosh; Cohn, Robert W; Sumanasekera, G U; Hewaparakrama, Kapila P

2008-01-01

Raman spectra of ordered carbon nanomaterials are quite sensitive to surface perturbations, including trace residues, structural defects and residual stress. This is demonstrated by a series of experiments with carbon nanotubes and graphene. Their spectra change due to subtle changes in preparation and attachment to the substrate and to each other. Differences are most clearly seen by forming a material into an air bridge and probing it in the air gap and at the anchor points. A monolayer graphene sheet, shows a larger disorder band at the anchor points than in the air gap. However, a bundle or rope of parallel-aligned single-wall nanotubes shows a larger disorder band in the gap than at the anchor points. For the graphene sheet the substrate surface deforms the graphene, leading to increases in the disorder band. For the rope, the close proximity of the nanotubes to each other appears to produce a larger stress than the rope resting on the substrate

AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.

Science.gov (United States)

Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W; Heinemann, Udo; Klussmann, Enno

2016-07-01

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Not all Anchors Weigh Equally.

Science.gov (United States)

Greenstein, Michael; Velazquez, Alexandra

2017-11-01

The anchoring bias is a reliable effect wherein a person's judgments are affected by initially presented information, but it is unknown specifically why this effect occurs. Research examining this bias suggests that elements of both numeric and semantic priming may be involved. To examine this, the present research used a phenomenon wherein people treat numeric information presented differently in Arabic numeral or verbal formats. We presented participants with one of many forms of an anchor that represented the same value (e.g., twelve hundred or 1,200). Thus, we could examine how a concept's meaning and its absolute numeric value affect anchoring. Experiments 1 and 2 showed that people respond to Arabic and verbal anchors differently. Experiment 3 showed that these differences occurred largely because people tend to think of numbers in digit format. This suggests that one's conceptual understanding of the anchored information matters more than its strict numeric value.

Investigation into the behaviour of concrete anchored diaphragm walls under earthquake condition

International Nuclear Information System (INIS)

Saba, H. R.; Rahaii, A. R.

2003-01-01

Diaphragm walls are frequently used in civil Engineering projects. Considering the variety and important volume of consumed materials (concrete, anchors and soil), one of the important factors for design and construction of these walls, are their behaviour under different executive, and loading conditions. In this paper, various models of concrete diaphragms with different number of anchors and soil parameters under static and dynamic loading have been investigated using finite element method with nonlinear models. Results including the internal forces in diaphragm walls, variation of forces in the anchors, shape of the sliding surface and variation of pressure in soil are obtained and compared. An experimental tool with suitable measurement systems for determining the pressure and internal forces was designed and realised. Also with similitude and dimensional analyses, diaphragms with different number of anchors were built and set on the shaking table test and experimented under different accelograms. Finally results of nonlinear dynamic analysis were compared with experimental results

MAIL1 is essential for development of the primary root but not of anchor roots.

Science.gov (United States)

Ühlken, Christine; Hoth, Stefan; Weingartner, Magdalena

2014-01-01

MAIN-LIKE1 (MAIL1) is a ubiquitously expressed nuclear protein, which has a crucial function during root development. We have recently described loss of function mutants for MAIL1, in which the organization and function of the primary root meristem is lost soon after germination. Moreover cell differentiation is impaired resulting in primary root growth arrest soon after emergence. Here we show that mail1 mutants form several anchor roots from the hypocotyl to root junction. These anchor roots show similar defects in the organization of the stem cell niche as the primary root. In contrast, differentiation processes are not impaired and thus anchor roots seem to be able to compensate for the loss of primary root function. Our data show that MAIL1 is essential for specification of cell fate in the primary root but not in anchor roots.

Dependence of optical phase modulation on anchoring strength of dielectric shield wall surfaces in small liquid crystal pixels

Science.gov (United States)

Isomae, Yoshitomo; Shibata, Yosei; Ishinabe, Takahiro; Fujikake, Hideo

2018-03-01

We demonstrated that the uniform phase modulation in a pixel can be realized by optimizing the anchoring strength on the walls and the wall width in the dielectric shield wall structure, which is the needed pixel structure for realizing a 1-µm-pitch optical phase modulator. The anchoring force degrades the uniformity of the phase modulation in ON-state pixels, but it also keeps liquid crystals from rotating against the leakage of an electric field. We clarified that the optimal wall width and anchoring strength are 250 nm and less than 10-4 J/m2, respectively.

Self-interacting polymer chains terminally anchored to adsorbing surfaces of three-dimensional fractal lattices

Science.gov (United States)

Živić, I.; Elezović-Hadžić, S.; Milošević, S.

2018-01-01

We have studied the adsorption problem of self-attracting linear polymers, modeled by self-avoiding walks (SAWs), situated on three-dimensional fractal structures, exemplified by 3d Sierpinski gasket (SG) family of fractals as containers of a poor solvent. Members of SG family are enumerated by an integer b (b ≥ 2), and it is assumed that one side of each SG fractal is an impenetrable adsorbing surface. We calculate the critical exponents γ1 ,γ11, and γs, which are related to the numbers of all possible SAWs with one, both, and no ends anchored to the adsorbing boundary, respectively. By applying the exact renormalization group (RG) method (for the first three members of the SG fractal family, b = 2 , 3, and 4), we have obtained specific values of these exponents, for θ-chain and globular polymer phase. We discuss their mutual relations and relations with corresponding values pertinent to extended polymer chain phase.

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

Energy Technology Data Exchange (ETDEWEB)

Kojima, Motoki; Akahoshi, Tomohiro; Okamoto, Kenji; Yanase, Hideshi [Tottori Univ. (Japan). Dept. of Chemistry and Biotechnology

2012-11-15

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae. (orig.)

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

Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

Science.gov (United States)

The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

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.

LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

DEFF Research Database (Denmark)

Parkyn, Celia J; Vermeulen, Esmeralda G M; Mootoosamy, Roy C

2008-01-01

The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly and consti......The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly...... required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrP(C) in biosynthetic compartments, with a concomitant lowering of surface PrP(C), suggesting that LRP1 expedites the trafficking of PrP(C) to the neuronal surface. PrP(C) and LRP1 can be co......-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrP(C) binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 microM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface...

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

DEFF Research Database (Denmark)

Schneider, Falk; Waithe, Dominic; Clausen, Mathias P

2017-01-01

(STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live cell plasma membrane and in actin cytoskeleton-free cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids......Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling, and they are suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy...... forming immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules, and highlight a powerful experimental approach to decipher specific influences on molecular plasma...

Identification of a peptide binding protein that plays a role in antigen presentation

International Nuclear Information System (INIS)

Lakey, E.K.; Margoliash, E.; Pierce, S.K.

1987-01-01

The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

Natural HLA-B*2705 Protein Ligands with Glutamine as Anchor Motif

Science.gov (United States)

Infantes, Susana; Lorente, Elena; Barnea, Eilon; Beer, Ilan; Barriga, Alejandro; Lasala, Fátima; Jiménez, Mercedes; Admon, Arie; López, Daniel

2013-01-01

The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705+ cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides. PMID:23430249

Liquifying PLDLLA Anchor Fixation in Achilles Reconstruction for Insertional Tendinopathy.

Science.gov (United States)

Boden, Stephanie A; Boden, Allison L; Mignemi, Danielle; Bariteau, Jason T

2018-04-01

Insertional Achilles tendinopathy (IAT) is a frequent cause of posterior heel pain and is often associated with Haglund's deformity. Surgical correction for refractory cases of IAT has been well studied; however, the method of tendon fixation to bone in these procedures remains controversial, and to date, no standard technique has been identified for tendon fixation in these surgeries. Often, after Haglund's resection, there is large exposed cancellous surface for Achilles reattachment, which may require unique fixation to optimize outcomes. Previous studies have consistently demonstrated improved patient outcomes after Achilles tendon reconstruction with early rehabilitation with protected weight bearing, evidencing the need for a strong and stable anchoring of the Achilles tendon that allows early weight bearing without tendon morbidity. In this report, we highlight the design, biomechanics, and surgical technique of Achilles tendon reconstruction with Haglund's deformity using a novel technique that utilizes ultrasonic energy to liquefy the suture anchor, allowing it to incorporate into surrounding bone. Biomechanical studies have demonstrated superior strength of the suture anchor utilizing this novel technique as compared with prior techniques. However, future research is needed to ensure that outcomes of this technique are favorable when compared with outcomes using traditional suture anchoring methods. Level V: Operative technique.

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

Science.gov (United States)

Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-10-04

Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach

The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis.

Science.gov (United States)

Deng, Simin; Xu, Tong; Fang, Qiong; Yu, Lei; Zhu, Jiaqi; Chen, Long; Liu, Jiahui; Zhou, Rui

2018-01-01

Streptococcus suis is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of S. suis has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in S. suis complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of sntA -deleted mutant strain Δ sntA are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ sntA . The abilities of anti-phagocytosis, survival in blood, and in vivo colonization of Δ sntA are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity via the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

Electrophoresis in ice surface grooves for probing protein affinity to a specific plane of ice crystal.

Science.gov (United States)

Inagawa, Arinori; Okada, Yusuke; Okada, Tetsuo

2018-06-01

Channel-like grooves are formed on the surface of frozen aqueous sucrose. They are filled with a freeze concentrated solution (FCS) and act as an efficient size-tunable separation field for micro and nanoparticles. The width of the channel can be easily varied by changing the temperature. Because the channel width decreases with decreasing temperature, particles become immobilized due to physical interference from the ice wall when the temperature reaches a threshold point specific to the particle size. Surface modification of particles can add a factor of chemical interaction between the particles and ice walls. In this study, anti-freeze proteins (AFPs) are anchored on 1µm-polystyrene (PS) particles, and their behavior in the surface grooves on the ice is studied. The threshold temperature is an effective criterion for evaluating chemical interactions between particles and ice walls. The AFP binding on 1µm PS particles lowers the threshold temperature by 2.5°C, indicating interactions between AFPs on the PS particles and the ice wall. Because the AFPs studied here show selectivity towards the prism plane, it is critical that the prism plane of the ice crystal is in contact with the FCS in the surface grooves. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Biomechanical comparison of traditional anchors to all-suture anchors in a double-row rotator cuff repair cadaver model.

Science.gov (United States)

Goschka, Andrew M; Hafer, Jason S; Reynolds, Kirk A; Aberle, Nicholas S; Baldini, Todd H; Hawkins, Monica J; McCarty, Eric C

2015-10-01

To further reduce the invasiveness of arthroscopic rotator cuff repair surgery the all-suture anchor has been developed. The all-suture anchor requires less bone removal and reduces the potential of loose body complications. The all-suture anchor must also have adequate biomechanical strength for the repair to heal. The hypothesis is there is no significant difference in the biomechanical performance of supraspinatus repairs using an all-suture anchor when compared to traditional solid-body suture anchors. Using nine shoulders per group, the supraspinatus tendon was dissected from the greater tuberosity. The four different double row repairs tested were (medial row/lateral row): A: ICONIX2/ICONIX2; B: ICONIX2/Stryker ReelX 3.9mm; C: ICONIX2/Stryker ReelX 4.5mm; D: Arthrex BioComposite CorkScrew FT 4.5mm/Arthrex BioComposite SwiveLock 4.75mm. The ICONIX2 was the only all-suture anchor tested. Tendons underwent cyclic loading from 10 to 100N for 500 cycles, followed by load-to-failure. Data was collected at cycles 5, 100, 200, 300, 400, and 500. One-way ANOVA analysis was used to assess significance (P≤0.05). The anchor combinations tested did not differ significantly in anterior (P>0.4) or posterior (P>0.3) gap formation, construct stiffness (P>0.7), ultimate load (P=0.06), or load to 5mm gap formation (P=0.84). The all-suture anchor demonstrated comparable biomechanical performance in multiple double-row anchor combinations to a combination of traditional solid-body anchors. Thus it may be an attractive option to further reduce the invasiveness of rotator cuff repairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

New 'one-step' method for the simultaneous synthesis and anchoring of organic monolith inside COC microchip channels

International Nuclear Information System (INIS)

Ladner, Yoann; Cretier, Gerard; Dugas, Vincent; Randon, Jerome; Faure, Karine; Bruchet, Anthony

2012-01-01

A new method for monolith synthesis and anchoring inside cyclic olefin copolymer (COC) microchannels in a single step is proposed. It is shown that type I photo-initiators, typically used in a polymerization mixture to generate free radicals during monolith synthesis, can simultaneously act as type II photo-initiators and react with the plastic surface through hydrogen abstraction. This mechanism is used to 'photo-graft' poly(ethylene glycol) methacrylate (PEGMA) on COC surfaces. Contact angle measurements were used to observe the changes in surface hydrophilicity when increasing initiator concentration and irradiation duration. The ability of type I photo-initiators to synthesize and anchor a monolith inside COC microchannels in a single step was proved through SEM observations. Different concentrations of photo-initiators were tried. Finally, electro-chromatographic separations of polycyclic aromatic hydrocarbons were realized to illustrate the beneficial effect of anchoring on chromatographic performances. The versatility of the method was demonstrated with two widely used photo-initiators: benzoin methyl ether (BME) and azobisisobutyronitrile (AIBN). (authors)

MAIL1 is essential for development of the primary root but not of anchor roots

OpenAIRE

Ühlken, Christine; Hoth, Stefan; Weingartner, Magdalena

2014-01-01

MAIN-LIKE1 (MAIL1) is a ubiquitously expressed nuclear protein, which has a crucial function during root development. We have recently described loss of function mutants for MAIL1, in which the organization and function of the primary root meristem is lost soon after germination. Moreover cell differentiation is impaired resulting in primary root growth arrest soon after emergence. Here we show that mail1 mutants form several anchor roots from the hypocotyl to root junction. These anchor root...

Non-uniformly functionalized titanium carbide-based MXenes as an anchoring material for Li-S batteries: A first-principles calculation

Science.gov (United States)

Sim, Eun Seob; Chung, Yong-Chae

2018-03-01

In this study, the influence of the non-uniform surface of F- and O-functionalized Ti2C on the anchoring behavior of lithium polysulfide (LiPS) is investigated using density functional theory. In order to consider the non-uniform surface, the substitutional, vacancy, and S-trapped sites of F- and O-functionalized Ti2C are designed. The anchoring behavior is investigated considering the adsorption energy of LiPS, reactivity between Li atoms and the substrate, and the reduction state of the S atoms. On the F-substitutional site of the O-functionalized surface, it is confirmed that the suppressing mechanism changes from the neutralization of S atoms to the anchoring of LiPS. However, too strong of an interaction between Ti atoms exposed at the vacancy site and S atoms induces trapping of the S atom at the vacancies of both F- and O-functionalized surfaces. As a result of the trapping of the S atom, the use of active material decreases. In addition, the S-trapped site originated from the vacancy site does not affect the suppressing mechanism. In conclusion, to optimize the Ti2C-based MXene as an anchoring material for Li-S batteries, the preparation process should be focused on eliminating the vacancy of functional groups.

Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

Directory of Open Access Journals (Sweden)

Mengya eNiu

2015-12-01

Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

Monogenean anchor morphometry: systematic value, phylogenetic signal, and evolution

Science.gov (United States)

Soo, Oi Yoon Michelle; Tan, Wooi Boon; Lim, Lee Hong Susan

2016-01-01

Background. Anchors are one of the important attachment appendages for monogenean parasites. Common descent and evolutionary processes have left their mark on anchor morphometry, in the form of patterns of shape and size variation useful for systematic and evolutionary studies. When combined with morphological and molecular data, analysis of anchor morphometry can potentially answer a wide range of biological questions. Materials and Methods. We used data from anchor morphometry, body size and morphology of 13 Ligophorus (Monogenea: Ancyrocephalidae) species infecting two marine mugilid (Teleostei: Mugilidae) fish hosts: Moolgarda buchanani (Bleeker) and Liza subviridis (Valenciennes) from Malaysia. Anchor shape and size data (n = 530) were generated using methods of geometric morphometrics. We used 28S rRNA, 18S rRNA, and ITS1 sequence data to infer a maximum likelihood phylogeny. We discriminated species using principal component and cluster analysis of shape data. Adams’s Kmult was used to detect phylogenetic signal in anchor shape. Phylogeny-correlated size and shape changes were investigated using continuous character mapping and directional statistics, respectively. We assessed morphological constraints in anchor morphometry using phylogenetic regression of anchor shape against body size and anchor size. Anchor morphological integration was studied using partial least squares method. The association between copulatory organ morphology and anchor shape and size in phylomorphospace was used to test the Rohde-Hobbs hypothesis. We created monogeneaGM, a new R package that integrates analyses of monogenean anchor geometric morphometric data with morphological and phylogenetic data. Results. We discriminated 12 of the 13 Ligophorus species using anchor shape data. Significant phylogenetic signal was detected in anchor shape. Thus, we discovered new morphological characters based on anchor shaft shape, the length between the inner root point and the outer root

Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

Science.gov (United States)

Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

2016-11-01

Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

Science.gov (United States)

Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

2016-02-01

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

The Holding Power of Anchors

Indian Academy of Sciences (India)

The efficiency of an anchor may be expressed as the ratio (holding force + weight of anchor). In dry sand .... the market at the beginning of the coming season in three sizes, namely 20, 35 and. 60 lb. These are ... Taylor frozen-flow hypothesis.

The establishment of Saccharomyces boulardii surface display system using a single expression vector.

Science.gov (United States)

Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

2014-03-01

In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

Science.gov (United States)

Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

2009-10-12

Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

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

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

78 FR 45104 - Model Manufactured Home Installation Standards: Ground Anchor Installations

Science.gov (United States)

2013-07-26

... anchor capacity. In granular soils, large lateral movements may produce failure planes that can reduce..., Consumer protection, Intergovernmental relations, Manufactured homes, Reporting and recordkeeping... relative to the plane of the ground surface and shall be permitted to be rounded to the nearest 5-degree...

Prion protein induced signaling cascades in monocytes

International Nuclear Information System (INIS)

Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, Ruediger; Vassallo, Neville; Herms, Jochen; Kretzschmar, Hans A.

2006-01-01

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP C ), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP C fusion proteins synthesized with a human Fc-tag. PrP C fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK 1,2 and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP C in monocytes and macrophages

Enhanced Charge Separation Efficiency in Pyridine-Anchored Phthalocyanine-Sensitized Solar Cells by Linker Elongation.

Science.gov (United States)

Ikeuchi, Takuro; Agrawal, Saurabh; Ezoe, Masayuki; Mori, Shogo; Kimura, Mutsumi

2015-11-01

A series of zinc phthalocyanine sensitizers (PcS22-24) having a pyridine anchoring group are designed and synthesized to investigate the structural dependence on performance in dye-sensitized solar cells. The pyridine-anchor zinc phthalocyanine sensitizer PcS23 shows 79 % incident-photon to current-conversion efficiency (IPCE) and 6.1 % energy conversion efficiency, which are comparable with similar phthalocyanine dyes having a carboxylic acid anchoring group. Based on DFT calculations, the high IPCE is attributed with the mixture of an excited-state molecular orbital of the sensitizer and the orbitals of TiO2 . Between pyridine and carboxylic acid anchor dyes, opposite trends are observed in the linker-length dependence of the IPCE. The red-absorbing PcS23 is applied for co-sensitization with a carboxyl-anchor organic dye D131 that has a complementary spectral response. The site-selective adsorption of PcS23 and D131 on the TiO2 surface results in a panchromatic photocurrent response for the whole visible-light region of sun light. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anchoring effects on early autobiographical memories.

Science.gov (United States)

Greenberg, Daniel L; Bishara, Anthony J; Mugayar-Baldocchi, Marino A

2017-10-01

Studies of childhood memory typically show that our earliest memories come from between three and four years of age. This finding is not universal, however. The age estimate varies across cultures and is affected by social influences. Research from the judgments and decision-making literature suggests that these estimates might also involve a judgment under uncertainty. Therefore, they might be susceptible to less social influences such as heuristics and biases. To investigate this possibility, we conducted two experiments that used anchoring paradigms to influence participants' estimates of their age during early autobiographical memories. In Experiment 1, participants answered either a high-anchor or a low-anchor question, and were warned that the anchor was uninformative; they went on to estimate their age during their earliest autobiographical memory. In Experiment 2, we replicated Experiment 1 and extended the design to examine additional early autobiographical memories. In both experiments, participants in the low-anchor condition gave earlier age estimates than those in the high-anchor condition. These results provide new insights into the methods used to investigate autobiographical memory. Moreover, they show that reports of early autobiographical memories can be influenced by a relatively light touch - a change to a single digit in a single question.

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.

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

The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

Science.gov (United States)

Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

2017-12-15

Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

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

An earth anchor system: installation and design guide.

Science.gov (United States)

R.L. Copstead; D.D. Studier

1990-01-01

A system for anchoring the guylines and skylines of cable yarding equipment is presented. A description of three types of tipping plate anchors is given. Descriptions of the installation equipment and methods specific to each type are given. Procedures for determining the correct number of anchors to install are included, as are guidelines for installing the anchors so...

Efficient production of D-tagatose using a food-grade surface display system.

Science.gov (United States)

Liu, Yi; Li, Sha; Xu, Hong; Wu, Lingtian; Xu, Zheng; Liu, Jing; Feng, Xiaohai

2014-07-16

D-tagatose, a functional sweetener, is commonly transformed from D-galactose by L-arabinose isomerase (L-AI). In this study, a novel type of biocatalyst, L-AI from Lactobacillus fermentum CGMCC2921 displayed on the spore surface of Bacillus subtilis 168, was developed for producing D-tagatose. The anchored L-AI, exhibiting the relatively high bioactivity, suggested that the surface display system using CotX as the anchoring protein was successfully constructed. The stability of the anchored L-AI was significantly improved. Specifically, the consolidation of thermal stability representing 87% of relative activity was retained even at 80 °C for 30 min, which remarkably favored the production of D-tagatose. Under the optimal conditions, the robust spores can convert 75% D-galactose (100 g/L) into D-tagatose after 24 h, and the conversion rate remained at 56% at the third cycle. Therefore, this biocatalysis system, which could express the target enzyme on the food-grade vector, was an alternative method for the value-added production of D-tagatose.

Using a biomimetic membrane surface experiment to investigate the activity of the magnetite biomineralisation protein Mms6† †Electronic supplementary information (ESI) available: Including Mms6 protein and peptide sequences, additional QCM-D and SEM data and protein modelling. See DOI: 10.1039/c5ra16469a Click here for additional data file.

Science.gov (United States)

Bird, Scott M.; Rawlings, Andrea E.; Galloway, Johanna M.

2016-01-01

Magnetotactic bacteria are able to synthesise precise nanoparticles of the iron oxide magnetite within their cells. These particles are formed in dedicated organelles termed magnetosomes. These lipid membrane compartments use a range of biomineralisation proteins to nucleate and regulate the magnetite crystallisation process. A key component is the membrane protein Mms6, which binds to iron ions and helps to control the formation of the inorganic core. We have previously used Mms6 on gold surfaces patterned with a self-assembled monolayer to successfully produce arrays of magnetic nanoparticles. Here we use this surface system as a mimic of the interior face of the magnetosome membrane to study differences between intact Mms6 and the acid-rich C-terminal peptide subregion of the Mms6 protein. When immobilised on surfaces, the peptide is unable to reproduce the particle size or homogeneity control exhibited by the full Mms6 protein in our experimental setup. Moreover, the peptide is unable to support anchoring of a dense array of nanoparticles to the surface. This system also allows us to deconvolute particle binding from particle nucleation, and shows that Mms6 particle binding is less efficient when supplied with preformed magnetite nanoparticles when compared to particles precipitated from solution in the presence of the surface immobilised Mms6. This suggests that Mms6 binds to iron ions rather than to magnetite surfaces in our system, and is perhaps a nucleating agent rather than a controller of magnetite crystal growth. The comparison between the peptide and the protein under identical experimental conditions indicates that the full length sequence is required to support the full function of Mms6 on surfaces. PMID:27019707

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.

Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

OpenAIRE

Åvall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-01-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactoc...

Improving performance by anchoring movement and "nerves".

Science.gov (United States)

Iso-Ahola, Seppo E; Dotson, Charles O; Jagodinsky, Adam E; Clark, Lily C; Smallwood, Lorraine L; Wilburn, Christopher; Weimar, Wendi H; Miller, Matthew W

2016-10-01

Golf's governing bodies' recent decision to ban all putting styles "anchoring one end of the club against the body" bridges an important practical problem with psychological theory. We report the first experiment testing whether anchoring provides technical and/or psychological advantage in competitive performance. Many "greats" of professional golf from Arnold Palmer and Jack Nicklaus to Tiger Woods have argued against anchoring, believing that it takes "nerves" out of competitive performance and therefore artificially levels the playing field. To shed more light on the issue, we tested participants' performance with anchored and unanchored putters under low and high pressure when controlling for the putter length. We found no statistically significant evidence for a technical advantage due to anchoring but a clear psychological advantage: participants who anchored their putters significantly outperformed unanchored counterparts under high, but not low, pressure. Results provide tentative evidence for the ban's justification from a competitive standpoint. However, before any definite conclusions can be made, more research is needed when using high-level golfers. Copyright © 2016 Elsevier B.V. All rights reserved.

Testing and modeling of cyclically loaded rock anchors

Directory of Open Access Journals (Sweden)

Joar Tistel

2017-12-01

Full Text Available The Norwegian Public Roads Administration (NPRA is planning for an upgrade of the E39 highway route at the westcoast of Norway. Fixed links shall replace ferries at seven fjord crossings. Wide spans and large depths at the crossings combined with challenging subsea topography and environmental loads call for an extension of existing practice. A variety of bridge concepts are evaluated in the feasibility study. The structures will experience significant loads from deadweight, traffic and environment. Anchoring of these forces is thus one of the challenges met in the project. Large-size subsea rock anchors are considered a viable alternative. These can be used for anchoring of floating structures but also with the purpose of increasing capacity of fixed structures. This paper presents first a thorough study of factors affecting rock anchor bond capacity. Laboratory testing of rock anchors subjected to cyclic loading is thereafter presented. Finally, the paper presents a model predicting the capacity of a rock anchor segment, in terms of a ribbed bar, subjected to a cyclic load history. The research assumes a failure mode occurring in the interface between the rock anchor and the surrounding grout. The constitutive behavior of the bonding interface is investigated for anchors subjected to cyclic one-way tensile loads. The model utilizes the static bond capacity curve as a basis, defining the ultimate bond τbu and the slip s1 at τbu. A limited number of input parameters are required to apply the model. The model defines the bond-slip behavior with the belonging rock anchor capacity depending on the cyclic load level (τmax cy/τbu, the cyclic load ratio (R = τmin cy/τmax cy, and the number of load cycles (N. The constitutive model is intended to model short anchor lengths representing an incremental length of a complete rock anchor.

PKA RIα/A-kinase anchoring proteins 10 signaling pathway and the prognosis of colorectal cancer.

Science.gov (United States)

Wang, Mojin; Li, Yuan; Wang, Rui; Wang, Ziqiang; Chen, Keling; Zhou, Bin; Zhou, Zongguang; Sun, Xiaofeng

2015-03-01

Previously study showed that the loss of the control of cAMP-dependent protein kinase A RIα (PKA RIα)/ A-kinase anchoring proteins 10 (AKAP10) signaling pathway initiate dysregulation of cellular healthy physiology leading to tumorigenesis. The aim of this study was to investigate the role of PKA RIα/AKAP10 signaling pathway in colorectal cancer (CRC). The AKAP10 expression at the mRNA and protein level have been analyzed in colon cancer cell lines, primary CRCs and matched normal mucosa samples, and compared in accordance with specific clinicopathological features of CRC. The correlation between expression of AKAP10 and PKA RIα were also analyzed. Compared with HCT116 and SW480 cells, the AKAP10 was significantly upregulated in the colon cell line KM12C and its metastatic counterparts, KM12SM and KM12L4A. Moreover, the KM12SM and KM12L4A having high metastatic potentials displayed the elevated levels of AKAP10 compared with KM12C having poor metastatic potential. A notably higher level of AKAP10 expression was found in CRC tissues at both mRNA and protein levels. Increased expression of AKAP10 in CRC patients was positively associated with the depth of invasion and the grade of differentiation. Univariate survival analysis showed that the increased expression of AKAP10 was related to poorer survival. Cox multivariate regression analysis confirmed that AKAP10 was an independent predictor of the overall survival of CRC patients. PKA RIα mRNA was also expressed at high levels in CRC. The correlation coefficient between mRNA expression of AKAP10 and PKA RIα in CRC was 0.417. AKAP10 mRNA overexpression was correlated significantly with PKA RIα. Our data indicated that PKA RIα/AKAP10 signaling pathway is associated with the progression and prognosis of CRC. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

Science.gov (United States)

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

Science.gov (United States)

Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Influence of Anchoring on Burial Depth of Submarine Pipelines.

Science.gov (United States)

Zhuang, Yuan; Li, Yang; Su, Wei

2016-01-01

Since the beginning of the twenty-first century, there has been widespread construction of submarine oil-gas transmission pipelines due to an increase in offshore oil exploration. Vessel anchoring operations are causing more damage to submarine pipelines due to shipping transportation also increasing. Therefore, it is essential that the influence of anchoring on the required burial depth of submarine pipelines is determined. In this paper, mathematical models for ordinary anchoring and emergency anchoring have been established to derive an anchor impact energy equation for each condition. The required effective burial depth for submarine pipelines has then been calculated via an energy absorption equation for the protection layer covering the submarine pipelines. Finally, the results of the model calculation have been verified by accident case analysis, and the impact of the anchoring height, anchoring water depth and the anchor weight on the required burial depth of submarine pipelines has been further analyzed.

Study on monitoring of deep foundation pit with SMW engineering method plus anchor cable retaining structure

Science.gov (United States)

Chen, Wenzhao; Cui, Wenping

2018-03-01

The SMW method has many advantages, such as little influence on the surrounding environment, good watertight performance, wide range of application, short construction period, low cost and so on. In this paper, we chose the SMW engineering method combined with anchor cable to support structure in the second phase deep foundation pit of Jinan LuJinDongCheng as the research object, monitored and analysed the horizontal displacement of the pile top, Peripheral surface subsidence and internal force of the anchor cable in the foundation pit. We also discussed the displacement, internal force of anchor cable and the settlement of surrounding environment in the excavation of foundation pit and in different stages of construction. Conclusion:(1)The maximum horizontal displacement of the retaining structure is closely related to the depth and time of excavation, the construction of anchor cable can well limit the deformation of pile body; (2)Groundwater seepage caused by foundation pit dewatering will change the effective stress of soil. The change of groundwater level has an important influence on the working behavior of smw anchor cable supporting structure.

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

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

Ringstone anchors from Gujarat, west coast of India

Digital Repository Service at National Institute of Oceanography (India)

Gaur, A.S.; Sundaresh; Tripati, S.; Bandodkar, S.N.

of Dwarka and Somanath have yielded several ringstone anchors along with other stone anchors such as triangular and grapnel types. The raw material used for these ring stones comprises basalt, sandstone and limestone. Earlier, these anchors were identified...

Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

International Nuclear Information System (INIS)

Sadeghi, H.; Da Silva, A.M.; Klein, C.

1988-01-01

The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

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.

Influence of Anchoring on Burial Depth of Submarine Pipelines.

Directory of Open Access Journals (Sweden)

Yuan Zhuang

Full Text Available Since the beginning of the twenty-first century, there has been widespread construction of submarine oil-gas transmission pipelines due to an increase in offshore oil exploration. Vessel anchoring operations are causing more damage to submarine pipelines due to shipping transportation also increasing. Therefore, it is essential that the influence of anchoring on the required burial depth of submarine pipelines is determined. In this paper, mathematical models for ordinary anchoring and emergency anchoring have been established to derive an anchor impact energy equation for each condition. The required effective burial depth for submarine pipelines has then been calculated via an energy absorption equation for the protection layer covering the submarine pipelines. Finally, the results of the model calculation have been verified by accident case analysis, and the impact of the anchoring height, anchoring water depth and the anchor weight on the required burial depth of submarine pipelines has been further analyzed.

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

Science.gov (United States)

Miller, Michelle M; Fogle, Jonathan E; Ross, Peter; Tompkins, Mary B

2013-04-01

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Silver nanoparticles anchored reduced graphene oxide for enhanced electrocatalytic activity towards methanol oxidation

Science.gov (United States)

Kumar, Sanjeev; Mahajan, Mani; Singh, Rajinder; Mahajan, Aman

2018-02-01

In this report, silver nanoparticles (Ag NPs) anchored reduced graphene oxide (rGO) sheets (rGO/Ag) nanohybrid has been explored as anode material in direct methanol fuel cells (DMFCs). The synthesized rGO/Ag nanohybrid is characterized by XRD, XPS, FTIR spectroscopy and HRTEM techniques. Cyclic voltammograms demonstrate that the rGO/Ag nanohybrid exhibits higher electrocatalytic activity in comparison to rGO sheets for methanol oxidation reaction (MOR). This enhancement is attributed to the synergetic effect produced by the presence of more active sites provided by Ag NPs anchored on a conducting network of large surface area rGO sheets.

Preparation and demonstration of poly(dopamine)-triggered attapulgite-anchored polyurethane as a high-performance rod-like elastomer to reinforce soy protein-isolated composites

Science.gov (United States)

Zhao, Shujun; Wen, Yingying; Wang, Zhong; Kang, Haijiao; Li, Jianzhang; Zhang, Shifeng; Ji, Yong

2018-06-01

Nanophase modification is an effective path to improve composite properties, however, it remains a great challenge to increase the mechanical strength of the modified materials without sacrificing elongation and toughness. This study presents a novel and efficient design for interface anchoring of a waterborne polyurethane (WPU) elastomer with attapulgite (ATP) triggered by poly(dopamine) (PDA) formation due to self-polymerization of the dopamine moieties. The WPU-PDA-ATP (WDA) rod-like elastomer served as an active enhancer for a soy protein isolate (SPI)-based composite to facilitate multiple interactions between SPI and the elastomer. As expected, the PDA layer was coated onto ATP, inducing the nanofiller to successfully anchor onto the WPU elastomer, as confirmed by solid-state 13C NMR, XPS, and ATR-FTIR results. Compared with the control SPI-based film, the tensile strength and toughness increased by 145.6% and 118.3% respectively by introducing WDA rod-like elastomer. The water resistance and thermal stability of the prepared SPI composites were also favorable. The proposed approach represents an efficient way to utilize high-performance elastomer in biobased materials to concurrently enhance strength and toughness.

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.

The Use of Comics-Based Cases in Anchored Instruction

Science.gov (United States)

Kneller, Matthew F.

2009-01-01

The primary purpose of this research was to understand how comics fulfill the role of anchor in an anchored instruction learning environment. Anchored instruction addresses the inert knowledge problem through the use of realistic multimedia stories, or "anchors," that embed a problem and the necessary data to solve it within the narrative. In the…

Effects of accuracy motivation and anchoring on metacomprehension judgment and accuracy.

Science.gov (United States)

Zhao, Qin

2012-01-01

The current research investigates how accuracy motivation impacts anchoring and adjustment in metacomprehension judgment and how accuracy motivation and anchoring affect metacomprehension accuracy. Participants were randomly assigned to one of six conditions produced by the between-subjects factorial design involving accuracy motivation (incentive or no) and peer performance anchor (95%, 55%, or no). Two studies showed that accuracy motivation did not impact anchoring bias, but the adjustment-from-anchor process occurred. Accuracy incentive increased anchor-judgment gap for the 95% anchor but not for the 55% anchor, which induced less certainty about the direction of adjustment. The findings offer support to the integrative theory of anchoring. Additionally, the two studies revealed a "power struggle" between accuracy motivation and anchoring in influencing metacomprehension accuracy. Accuracy motivation could improve metacomprehension accuracy in spite of anchoring effect, but if anchoring effect is too strong, it could overpower the motivation effect. The implications of the findings were discussed.

Dynamic performance of concrete undercut anchors for Nuclear Power Plants

Energy Technology Data Exchange (ETDEWEB)

Mahrenholtz, Christoph, E-mail: christoph@mahrenholtz.net; Eligehausen, Rolf

2013-12-15

Graphical abstract: - Highlights: • Behavior of undercut anchors under dynamic actions simulating earthquakes. • First high frequency load and crack cycling tests on installed concrete anchors ever. • Comprehensive review of anchor qualification for Nuclear Power Plants. - Abstract: Post-installed anchors are widely used for structural and nonstructural connections to concrete. In many countries, concrete anchors used for Nuclear Power Plants have to be qualified to ensure reliable behavior even under extreme conditions. The tests required for qualification of concrete anchors are carried out at quasi-static loading rates well below the rates to be expected for dynamic actions deriving from earthquakes, airplane impacts or explosions. To investigate potentially beneficial effects of high loading rates and cycling frequencies, performance tests on installed undercut anchors were conducted. After introductory notes on anchor technology and a comprehensive literature review, this paper discusses the qualification of anchors for Nuclear Power Plants and the testing carried out to quantify experimentally the effects of dynamic actions on the load–displacement behavior of undercut anchors.

Association of functional genetic variants of A-kinase anchoring protein 10 with QT interval length in full-term Polish newborns.

Science.gov (United States)

Łoniewska, Beata; Kaczmarczyk, Mariusz; Clark, Jeremy Simon; Gorący, Iwona; Horodnicka-Józwa, Anita; Ciechanowicz, Andrzej

2015-03-16

A-Kinase Anchoring Proteins (AKAPs) coordinate the specificity of protein kinase A signaling by localizing the kinase to subcellular sites. The 1936G (V646) AKAP10 allele has been associated in adults with low cholinergic/vagus nerve sensitivity, shortened PR intervals in ECG recording and in newborns with increased blood pressure and higher cholesterol cord blood concentration. The aim of the study was to answer the question of whether 1936A > G AKAP10 polymorphism is associated with the newborn electrocardiographic variables. Electrocardiograms were recorded from 114 consecutive healthy Polish newborns (55 females, 59 males), born after 37 gestational weeks to healthy women with uncomplicated pregnancies. All recordings were made between 3(rd) and 7(th) day of life to avoid QT variability. The heart rate per minute and duration of PR, QRS, RR and QT intervals were usually measured. The ECGs were evaluated independently by three observers. At birth, cord blood of neonates was obtained for isolation of genomic DNA. The distribution of anthropometric and electrocardiographic variables in our cohort approached normality (skewness G variant and QTc interval in Polish newborns.

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.

Additional Haptic Information Provided by Anchors Reduces Postural Sway in Young Adults Less Than Does Light Touch

Directory of Open Access Journals (Sweden)

Renato Moraes

2018-06-01

Full Text Available This study investigated the effect of adding haptic information to the control of posture, as well as comparing the effect of both the “light touch” (LT and “anchor system” (AS paradigms on postural sway. Additionally, it compared the effect of location and number of points of contact to the control of posture in young adults. The location consisted of using the anchors tied to the finger and held by the hands, and, for LT, the fingertip. For the number of points of contact, participants used two hands, and then separately the dominant hand, and the non-dominant hand, for both anchor and LT paradigms. Participants stood upright with feet-together and in tandem position while performing tasks that combined the use of anchors and LT, points of contact (hand grip and finger, and number of points of contact (two hands and one hand. In this study, the anchors consist of holding in each hand a flexible cable with the other end attached to the ground. The LT consists of slightly touching a rigid surface with the tip of the index finger. The results showed, first, that the anchors improved postural control less than did the LT. Second, they revealed that holding the anchors with the hands or with them tied to the fingertip resulted in a similar reduction in postural sway only in the tandem position. For the feet-together position, the anchors tied to the fingertip were ineffective. Similarly, the use of one or two hands did not affect the contribution of the anchors. However, using two hands in the LT condition was more effective than was one hand. Third, our results showed the presence of a temporal delay between force and center-of-pressure (COP for the anchors, only in the AP direction with feet-together. In conclusion, overall, the anchors were less effective in reducing postural sway than was the LT. The anchors attached to fingertips were as effective as the hand-held anchors in the tandem position, yet ineffective during foot

End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

Science.gov (United States)

Whitmore, Mark D; Grest, Gary S; Douglas, Jack F; Kent, Michael S; Suo, Tongchuan

2016-11-07

An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ 1/3 , for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

Studies on the mechanical behavior of rock anchors. ; Results of in-situ pull-out tests. Rock anchor no rikigaku kyodo ni kansuru kenkyu. ; Gen prime ichi shiken ni okeru anchor no kyodo

Energy Technology Data Exchange (ETDEWEB)

Arimoto, K.; Ebisu, S.; Nakagawa, M.; Usui, M.; Someya, T.; Machida, N. (Okumura Corp., Tokyo (Japan))

1991-10-31

The rock anchor method is planned to apply to some permanent structures but since this method was developed for temporary structures, the clarification of the transferring mechanism of force from an anchor to a rockmass, the fracture mechanism and the development of the dynamic model have not been established. This paper arranged the data obtained by a full-scale, in-situ pulling out test of a rock anchor as the first step to understand the dynamic behavior and analyzed by paying attetion to the modulus of deformation of the rockmass where the anchor was embedded to elucidate the affecting degree of rockmass modulus of deformation, the embedded length and the tendon diameter on the dynamic behavior of the anchor. The rock anchor behavior could be expressed accurately by applying a theoretical solution derived by the balancing condition of forces at the boundary face. Especially, when the rockmass is uniform and the fracture occurrs at the interface between the tendon and grout, this approach can express the fracture with the accuracy similar to that made by the finite element method. 6 refs., 11 figs.,1 tab.

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.

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.

Containment liner plate anchors and steel embedments test results

International Nuclear Information System (INIS)

Chang-Lo, P.L.; Johnson, T.E.; Pfeifer, B.W.

1977-01-01

This paper summarizes test data on shear load and deformation capabilities for liner plate line anchors and structural steel embedments in reinforced and prestressed concrete nuclear containments. Reinforced and prestressed nuclear containments designed and constructed in the United States are lined with a minimum of 0.64 cm steel plate. The liner plates are anchored by the use of either studs or structural members (line anchors) which usually run in the vertical direction. This paper will only address line anchors. Static load versus displacement test data is necessary to assure that the design is adequate for the maximum loads. The test program for the liner anchors had the following major objectives: determine load versus displacement data for a variety of anchors considering structural tees and small beams with different weld configurations, from the preceding tests, determine which anchors would lead to an economical and extremely safe design and test these anchors for cyclic loads resulting from thermal fluctuations. Various concrete embeds in the containment and other structures are subjected to loads such as pipe rupture which results in shear. Since many of the loads are transient by nature, it is necessary to know the load-displacement relationship so that the energy absorption can be determined. The test program for the embeds had the following objectives: determine load-displacement relationship for various size anchors from 6.5 cm 2 to 26 cm 2 with maximum capacities of approximately 650 kN; determine the effect of various anchor width-to-thickness ratios for the same shear area

Modified Kidner procedure utilizing a Mitek bone anchor.

Science.gov (United States)

Dawson, D M; Julsrud, M E; Erdmann, B B; Jacobs, P M; Ringstrom, J B

1998-01-01

The recent development of small bone suture anchors has created several potential applications in reconstructive surgery of the foot. Mitek bone anchors are simple to insert, require less aggressive dissection and surgical time than reefing of the redundant posterior tibial tendon, and are a reliable method of tendon-to-bone fixation. Mitek bone anchors are an excellent technique for the treatment of redundant tibialis posterior tendon following a modified Kidner procedure. In modified Kidner procedures involving an excessively large os tibiale externum, Mitek anchoring of the redundant tibialis posterior tendon to the navicular bone is an excellent means for secure plication of the posterior tibial tendon in cases involving intraoperative tendon laxity. A description of the Mitek Anchor System and technique of application in a modified Kinder procedure is presented. The purpose of this study was to describe patient satisfaction and long-term clinical outcomes of the modified Kinder procedure with and without the Mitek bone anchoring system. A retrospective study of the modified Kinder procedure was performed with 13 patients being evaluated, seven with Mitek anchoring and six without. The University of Maryland 100-point Painful Foot Center Scoring System was modified to be more specific to the modified Kinder procedure for assessment of subjective long-term results. Patient overall satisfaction was rated good to excellent by 85.6% of patients in the Mitek group and by 100% of patients in the non-Mitek group. Use of the Mitek anchor allowed for quicker postoperative recovery to resumption of ambulation without assistive devices (average of 3 weeks vs. 4.42 weeks) and a quicker return to pain-free ambulation in normal shoegear (average of 4 weeks vs. 6 weeks). Mitek anchoring of the tibialis posterior tendon, theoretically, increases medial arch support as evidenced by 14% of the Mitek group and 67% of the non-Mitek group requiring postoperative orthotics.

Editorial Commentary: All-Suture Anchors, Foam Blocks, and Biomechanical Testing.

Science.gov (United States)

Brand, Jefferson C

2017-06-01

Barber's biomechanical work is well known to Arthroscopy's readers as thorough, comprehensive, and inclusive of new designs as they become available. In "All-Suture Anchors: Biomechanical Analysis of Pullout Strength, Displacement, and Failure Mode," the latest iteration, Barber and Herbert test all-suture anchors in both porcine femurs and biphasic foam. While we await in vivo clinical trials that compare all-suture anchors to currently used anchors, Barber and Herbert have provided data to inform anchor choice, and using their biomechanical data at time zero from all-suture anchor trials in an animal model, we can determine the anchors' feasibility for human clinical investigations. Copyright © 2017 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Spectroscopic and first principles investigation on 4-[(4-pyridinylmethylene)amino]-benzoic acid bearing pyridyl and carboxyl anchoring groups

Science.gov (United States)

Zhang, Lei; Wang, Qiaoyi

2018-03-01

We report a combined experimental and computational investigation on the structure and photophysics of 4-[(4-pyridinylmethylene)amino]-benzoic acid, a functional molecule bearing two anchoring groups for attachment onto a TiO2 surface and perovskite surface, for potential solar cell application. This molecule possesses interesting adsorption properties in perovskite solar cell because the pyridyl group serves as the Lewis base and targets Lewis acidic sites in the perovskite surface, while the carboxyl group targets TiO2 surface, improving the coupling between the perovskite surface and the TiO2 surface. The electronic structures of the molecule and its photochemistry are revealed by the UV-vis absorption spectra and the fluorescence spectra under visible light irradiation, which are combined with density functional theory (DFT) and time-dependent density functional theory (TDDFT) analysis. Considering the bi-anchoring groups and the conjugated π system embedded in the molecule, we anticipate it can molecular engineer the TiO2/perovskite interface in perovskite solar cell.

Self-Assembled Monolayers on Gold of β-Cyclodextrin Adsorbates with Different Anchoring Groups

NARCIS (Netherlands)

Méndez Ardoy, Alejandro; Steentjes, Tom; Kudernac, Tibor; Huskens, Jurriaan

2014-01-01

We designed multivalent β-cyclodextrin-based adsorbates bearing different anchoring groups aiming to yield stable monolayers with improved packing and close contact of the cavity to the gold surface. Toward this end the primary rim of the β-cyclodextrin was decorated with several functional groups,

Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

Directory of Open Access Journals (Sweden)

Ma G

2013-03-01

Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

Relation between anchorings of liquid crystals and conformation changes in aligning agents by the Langmuir-Blodgett film technique investigation

International Nuclear Information System (INIS)

Zhu, Y.; Lu, Z.; Wei, Y.

1995-01-01

The anchoring direction of liquid crystals on a solid substrate surface depends upon many parameters characterizing the liquid-crystal--substrate interface, a variation of which may change this anchoring direction leading to the so-called anchoring transition. Here, based on the Langmuir-Blodgett film technique, we present two model systems to study the relation between anchoring directions and the conformation changes in aligning agents. A double-armed crown ether liquid crystal and a side chain polymer liquid crystal at an air-water interface both show phase transitions, accompanied by conformation changes. However, when the monolayers in different phases were transferred onto solid substrates to orient liquid crystals, we found that for the crown ether material the conformation change can alter the anchoring of liquid crystals between homeotropic and homogeneous alignments, while for the polymer liquid crystal, despite the conformation changes, the liquid crystals can only be aligned homeotropically. The involved mechanisms were briefly discussed in terms of the Landau-type phenomenological theory

The GPI-anchored protein Ecm33 is vital for conidiation, cell wall integrity, and multi-stress tolerance of two filamentous entomopathogens but not for virulence.

Science.gov (United States)

Chen, Ying; Zhu, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

2014-06-01

Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in ΔBbecm33 (76 %) than in ΔMrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in ΔBbecm33 only. Consequently, ΔBbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than ΔMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca(2+) during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in ΔBbecm33 but was unaffected in ΔMrecm33, unlike a similar decrease (25-28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither ΔBbecm33 nor ΔMrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

Energy Technology Data Exchange (ETDEWEB)

Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

2012-03-01

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

International Nuclear Information System (INIS)

Du, Yijun; Pattnaik, Asit K.; Song, Cheng; Yoo, Dongwan; Li, Gang

2012-01-01

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide–anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

Protein design on computers. Five new proteins: Shpilka, Grendel, Fingerclasp, Leather, and Aida.

Science.gov (United States)

Sander, C; Vriend, G; Bazan, F; Horovitz, A; Nakamura, H; Ribas, L; Finkelstein, A V; Lockhart, A; Merkl, R; Perry, L J

1992-02-01

What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded beta-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Finger-clasp, a dimer of interdigitating beta-beta-alpha units, the simplest variant of the "handshake" structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather--a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.

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

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.

Outcomes of the modified Brostrom procedure using suture anchors for chronic lateral ankle instability--a prospective, randomized comparison between single and double suture anchors.

Science.gov (United States)

Cho, Byung-Ki; Kim, Yong-Min; Kim, Dong-Soo; Choi, Eui-Sung; Shon, Hyun-Chul; Park, Kyoung-Jin

2013-01-01

The present prospective, randomized study was conducted to compare the clinical outcomes of the modified Brostrom procedure using single and double suture anchors for chronic lateral ankle instability. A total of 50 patients were followed up for more than 2 years after undergoing the modified Brostrom procedure. Of the 50 procedures, 25 each were performed using single and double suture anchors by 1 surgeon. The Karlsson scale had improved significantly to 89.8 points and 90.6 points in the single and double anchor groups, respectively. Using the Sefton grading system, 23 cases (92%) in the single anchor group and 22 (88%) in the double anchor group achieved satisfactory results. The talar tilt angle and anterior talar translation on stress radiographs using the Telos device had improved significantly to an average of 5.7° and 4.6 mm in the single anchor group and 4.5° and 4.3 mm in the double anchor group, respectively. The double anchor technique was superior with respect to the postoperative talar tilt. The single and double suture anchor techniques produced similar clinical and functional outcomes, with the exception of talar tilt as a reference of mechanical stability. The modified Brostrom procedure using both single and double suture anchors appears to be an effective treatment method for chronic lateral ankle instability. Copyright © 2013 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

Surface-Exposed Lipoproteins: An Emerging Secretion Phenomenon in Gram-Negative Bacteria.

Science.gov (United States)

Wilson, Marlena M; Bernstein, Harris D

2016-03-01

Bacterial lipoproteins are hydrophilic proteins that are anchored to a cell membrane by N-terminally linked fatty acids. It is widely believed that nearly all lipoproteins produced by Gram-negative bacteria are either retained in the inner membrane (IM) or transferred to the inner leaflet of the outer membrane (OM). Lipoproteins that are exposed on the cell surface have also been reported but are generally considered to be rare. Results from a variety of recent studies, however, now suggest that the prevalence of surface-exposed lipoproteins has been underestimated. In this review we describe the evidence that the surface exposure of lipoproteins in Gram-negative bacteria is a widespread phenomenon and discuss possible mechanisms by which these proteins might be transported across the OM. Published by Elsevier Ltd.

Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

Directory of Open Access Journals (Sweden)

Jun Ishii

Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

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

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.

Understanding the low uptake of bone-anchored hearing aids: a review.

Science.gov (United States)

Powell, R; Wearden, A; Pardesi, S M; Green, K

2017-03-01

Bone-anchored hearing aids improve hearing for patients for whom conventional behind-the-ear aids are problematic. However, uptake of bone-anchored hearing aids is low and it is important to understand why this is the case. A narrative review was conducted. Studies examining why people accept or decline bone-anchored hearing aids and satisfaction levels of people with bone-anchored hearing aids were reviewed. Reasons for declining bone-anchored hearing aids included limited perceived benefits, concerns about surgery, aesthetic concerns and treatment cost. No studies providing in-depth analysis of the reasons for declining or accepting bone-anchored hearing aids were identified. Studies of patient satisfaction showed that most participants reported benefits with bone-anchored hearing aids. However, most studies used cross-sectional and/or retrospective designs and only included people with bone-anchored hearing aids. Important avenues for further research are in-depth qualitative research designed to fully understand the decision-making process for bone-anchored hearing aids and rigorous quantitative research comparing satisfaction of people who receive bone-anchored hearing aids with those who receive alternative (or no) treatments.

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

Career anchors and learning plan (part one

Directory of Open Access Journals (Sweden)

Daniela Brečko

2006-12-01

Full Text Available The article is divided into three parts. The first part concentrates on how important career is for an individual, organization and society. The author establishes that understanding of career has changed dramatically and does not only refer to climbing up the career ladder, but also moving off or even down the career ladder. The notion of career, as a lifelong and professional path, encompasses all aspects of human personality and their roles acquired through one's life. On basis of vast and longitudinal research, where the author has studied career anchors of individuals, it is the objective of the author to find out on basis of what grounds do the individuals decide to take certain directions in their careers and how learning contributes to such decisions. As a source the author has used Shein's theory of career anchors. Part one describes in greater detail 8 different career anchors and introduces their main features with the findings of the research, which refer to the analysis of professions (work positions and established career anchors. The author thus verifies the hypothesis that career anchors do exist in our area.

Self-tapping ability of carbon fibre reinforced polyetheretherketone suture anchors.

Science.gov (United States)

Feerick, Emer M; Wilson, Joanne; Jarman-Smith, Marcus; Ó'Brádaigh, Conchur M; McGarry, J Patrick

2014-10-01

An experimental and computational investigation of the self-tapping ability of carbon fibre reinforced polyetheretherketone (CFR-PEEK) has been conducted. Six CFR-PEEK suture anchor designs were investigated using PEEK-OPTIMA® Reinforced, a medical grade of CFR-PEEK. Experimental tests were conducted to investigate the maximum axial force and torque required for self-taping insertion of each anchor design. Additional experimental tests were conducted for some anchor designs using pilot holes. Computational simulations were conducted to determine the maximum stress in each anchor design at various stages of insertion. Simulations also were performed to investigate the effect of wall thickness in the anchor head. The maximum axial force required to insert a self-tapping CFR-PEEK suture anchor did not exceed 150 N for any anchor design. The maximum torque required to insert a self-tapping CFR-PEEK suture anchor did not exceed 0.8 Nm. Computational simulations reveal significant stress concentrations in the region of the anchor tip, demonstrating that a re-design of the tip geometry should be performed to avoid fracture during self-tapping, as observed in the experimental component of this study. This study demonstrates the ability of PEEK-OPTIMA Reinforced suture anchors to self-tap polyurethane foam bone analogue. This provides motivation to further investigate the self-tapping ability of CFR-PEEK suture anchors in animal/cadaveric bone. An optimised design for CFR-PEEK suture anchors offers the advantages of radiolucency, and mechanical properties similar to bone with the ability to self-tap. This may have positive implications for reducing surgery times and the associated costs with the procedure. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Career anchors and values from different career management perspectives

Directory of Open Access Journals (Sweden)

Rodrigo Cunha da Silva

2016-06-01

Full Text Available Purpose – To analyze the relationships between career anchors and young Generation Y professionals’ values, from the career concept perspective. Design/methodology/approach – Research concerning the proposed objective was carried out through quantitative research involving 189 Business Administration majors from a Catholic university in São Paulo, Brazil. We used two instruments to identify the career anchors and values of respondents: Schein (1990 and Schwartz (1994, respectively. We used statistical techniques to explore the relationships between career anchors and values. Findings – Among the results, mention should be made to the statistical relationships found between analyzed career anchors and values. It is also important to stress that, although the Lifestyle career anchor was predominantly present in the conglomerate division, this anchor was the predominant characteristic in the differentiation of the smaller group of respondents, the new career group. The General Management Career Anchor, which presents a lower incidence, is the predominant characteristic of the larger group, referring to organizational careers. As well as the Lifestyle career anchor, the Hedonism value was predominant among respondents. Originality/value – The need to consider the following was found: Generation Y presents generational characteristics that drive people management to propose work structures that offer activities to generate learning, pleasure, self-fulfillment and conciliation between work and personal life.

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.

Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

Science.gov (United States)

Chen, Xianzhong

2017-03-04

The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

Test Score Equating Using Discrete Anchor Items versus Passage-Based Anchor Items: A Case Study Using "SAT"® Data. Research Report. ETS RR-14-14

Science.gov (United States)

Liu, Jinghua; Zu, Jiyun; Curley, Edward; Carey, Jill

2014-01-01

The purpose of this study is to investigate the impact of discrete anchor items versus passage-based anchor items on observed score equating using empirical data.This study compares an "SAT"® critical reading anchor that contains more discrete items proportionally, compared to the total tests to be equated, to another anchor that…

On Moderator Detection in Anchoring Research: Implications of Ignoring Estimate Direction

Directory of Open Access Journals (Sweden)

Nathan N. Cheek

2018-05-01

Full Text Available Anchoring, whereby judgments assimilate to previously considered standards, is one of the most reliable effects in psychology. In the last decade, researchers have become increasingly interested in identifying moderators of anchoring effects. We argue that a drawback of traditional moderator analyses in the standard anchoring paradigm is that they ignore estimate direction—whether participants’ estimates are higher or lower than the anchor value. We suggest that failing to consider estimate direction can sometimes obscure moderation in anchoring tasks, and discuss three potential analytic solutions that take estimate direction into account. Understanding moderators of anchoring effects is essential for a basic understanding of anchoring and for applied research on reducing the influence of anchoring in real-world judgments. Considering estimate direction reduces the risk of failing to detect moderation.

The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

Directory of Open Access Journals (Sweden)

Ravikrishna Ramanujam

Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

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

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.

Biased calculations: Numeric anchors influence answers to math equations

Directory of Open Access Journals (Sweden)

Andrew R. Smith

2011-02-01

Full Text Available People must often perform calculations in order to produce a numeric estimate (e.g., a grocery-store shopper estimating the total price of his or her shopping cart contents. The current studies were designed to test whether estimates based on calculations are influenced by comparisons with irrelevant anchors. Previous research has demonstrated that estimates across a wide range of contexts assimilate toward anchors, but none has examined estimates based on calculations. In two studies, we had participants compare the answers to math problems with anchors. In both studies, participants' estimates assimilated toward the anchor values. This effect was moderated by time limit such that the anchoring effects were larger when the participants' ability to engage in calculations was limited by a restrictive time limit.

Technical Note: Drifting vs. anchored flux chambers for measuring greenhouse gas emissions from running waters

Science.gov (United States)

Lorke, A.; Bodmer, P.; Noss, C.; Alshboul, Z.; Koschorreck, M.; Somlai, C.; Bastviken, D.; Flury, S.; McGinnis, D. F.; Maeck, A.; Müller, D.; Premke, K.

2015-09-01

Stream networks were recently discovered as major but poorly constrained natural greenhouse gas (GHG) sources. A fundamental problem is that several measurement approaches have been used without cross comparisons. Flux chambers represent a potentially powerful methodological approach if robust and reliable ways to use chambers on running water can be defined. Here we compare the use of anchored and freely drifting chambers on various streams having different flow velocities. The study clearly shows that (1) drifting chambers have a very small impact on the water turbulence under the chamber and thus generate more reliable fluxes, (2) anchored chambers enhance turbulence under the chambers and thus elevate fluxes, (3) the bias of the anchored chambers greatly depends on chamber design and sampling conditions, and (4) there is a promising method to reduce the bias from anchored chambers by using a flexible plastic foil seal to the water surface rather than having rigid chamber walls penetrating into the water. Altogether, these results provide novel guidance on how to apply flux chambers in running water, which will have important consequences for measurements to constrain the global GHG balances.

Technical note: drifting versus anchored flux chambers for measuring greenhouse gas emissions from running waters

Science.gov (United States)

Lorke, A.; Bodmer, P.; Noss, C.; Alshboul, Z.; Koschorreck, M.; Somlai-Haase, C.; Bastviken, D.; Flury, S.; McGinnis, D. F.; Maeck, A.; Müller, D.; Premke, K.

2015-12-01

Stream networks have recently been discovered to be major but poorly constrained natural greenhouse gas (GHG) sources. A fundamental problem is that several measurement approaches have been used without cross-comparisons. Flux chambers represent a potentially powerful methodological approach if robust and reliable ways to use chambers on running water can be defined. Here we compare the use of anchored and freely drifting chambers on various streams with different flow velocities. The study clearly shows that (1) anchored chambers enhance turbulence under the chambers and thus elevate fluxes, (2) drifting chambers have a very small impact on the water turbulence under the chamber and thus generate more reliable fluxes, (3) the bias of the anchored chambers greatly depends on chamber design and sampling conditions, and (4) there is a promising method to reduce the bias from anchored chambers by using a flexible plastic foil collar to seal the chambers to the water surface, rather than having rigid chamber walls penetrating into the water. Altogether, these results provide novel guidance on how to apply flux chambers in running water, which will have important consequences for measurements to constrain the global GHG balances.

Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

Directory of Open Access Journals (Sweden)

Anton R Dluzewski

2008-08-01

Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

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.

Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination

Energy Technology Data Exchange (ETDEWEB)

Kobashigawa, Yoshihiro; Saio, Tomohide [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan); Ushio, Masahiro [Hokkaido University, Graduate School of Life Science (Japan); Sekiguchi, Mitsuhiro [Astellas Pharma Inc., Analysis and Pharmacokinetics Research Labs, Department of Drug Discovery (Japan); Yokochi, Masashi; Ogura, Kenji; Inagaki, Fuyuhiko, E-mail: finagaki@pharm.hokudai.ac.jp [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan)

2012-05-15

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

Laser-Modified Surface Enhances Osseointegration and Biomechanical Anchorage of Commercially Pure Titanium Implants for Bone-Anchored Hearing Systems

Science.gov (United States)

Omar, Omar; Simonsson, Hanna; Palmquist, Anders; Thomsen, Peter

2016-01-01

Osseointegrated implants inserted in the temporal bone are a vital component of bone-anchored hearing systems (BAHS). Despite low implant failure levels, early loading protocols and simplified procedures necessitate the application of implants which promote bone formation, bone bonding and biomechanical stability. Here, screw-shaped, commercially pure titanium implants were selectively laser ablated within the thread valley using an Nd:YAG laser to produce a microtopography with a superimposed nanotexture and a thickened surface oxide layer. State-of-the-art machined implants served as controls. After eight weeks’ implantation in rabbit tibiae, resonance frequency analysis (RFA) values increased from insertion to retrieval for both implant types, while removal torque (RTQ) measurements showed 153% higher biomechanical anchorage of the laser-modified implants. Comparably high bone area (BA) and bone-implant contact (BIC) were recorded for both implant types but with distinctly different failure patterns following biomechanical testing. Fracture lines appeared within the bone ~30–50 μm from the laser-modified surface, while separation occurred at the bone-implant interface for the machined surface. Strong correlations were found between RTQ and BIC and between RFA at retrieval and BA. In the endosteal threads, where all the bone had formed de novo, the extracellular matrix composition, the mineralised bone area and osteocyte densities were comparable for the two types of implant. Using resin cast etching, osteocyte canaliculi were observed directly approaching the laser-modified implant surface. Transmission electron microscopy showed canaliculi in close proximity to the laser-modified surface, in addition to a highly ordered arrangement of collagen fibrils aligned parallel to the implant surface contour. It is concluded that the physico-chemical surface properties of laser-modified surfaces (thicker oxide, micro- and nanoscale texture) promote bone bonding

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.

76 FR 30301 - Commercial Acquisition; Anchor Tenancy

Science.gov (United States)

2011-05-25

... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION 48 CFR Part 1812 RIN 2700-AD64 Commercial... consistent with NASA's authority under Section 401 of the Commercial Space Competitiveness Act (CSCA) of 1992. NASA may enter into multi-year anchor tenancy contracts for commercial space goods or services. Anchor...

A comparison of lateral ankle ligament suture anchor strength.

Science.gov (United States)

Barber, F Alan; Herbert, Morley A; Crates, John M

2013-06-01

Lateral ankle ligament repairs increasingly use suture anchors instead of bone tunnels. Our purpose was to compare the biomechanical properties of a knotted and knotless suture anchor appropriate for a lateral ankle ligament reconstruction. In porcine distal fibulae, 10 samples of 2 different PEEK anchors were inserted. The attached sutures were cyclically loaded between 10N and 60N for 200 cycles. A destructive pull was performed and failure loads, cyclic displacement, stiffness, and failure mode recorded. PushLock 2.5 anchors failed before 200 cycles. PushLock 100 cycle displacement was less than Morphix 2.5 displacement (panchors completing 200 cycles was 86.5N (PushLock) and 252.1N (Morphix) (panchor breaking and suture breakage. The knotted Morphix demonstrated more displacement and greater failure strength than the knotless PushLock. The PushLock failed consistently with suture breaking. The Morphix anchor failed both by anchor breaking and by suture breaking. Copyright © 2012 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.

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...

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.

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.

Post-installed concrete anchors in nuclear power plants: Performance and qualification

International Nuclear Information System (INIS)

Mahrenholtz, Philipp; Eligehausen, Rolf

2015-01-01

Graphical abstract: - Highlights: • Review of qualification and design regulations for anchors in nuclear power plants. • First complete set of nuclear anchor load–displacement data and its evaluation ever. • Demonstration of robust test behavior of a qualified post-installed anchor product. - Abstract: In nuclear power plants (NPPs), post-installed anchors are widely used for structural and non-structural connections to concrete. In many countries, anchor products employed for safety relevant applications have to be approved by the authorities. For the high safety standards in force for NPPs, special requirements have to be met to allow for extreme design situations. This paper presents an experimental test program conducted to evaluate the performance of anchors according to the German Guideline for Anchorages in Nuclear Power Plants and Nuclear Technology Installations (DIBt KKW Leitfaden, 2010). After a brief introduction to anchor behavior and the regulative context, the results of tension and shear tests carried out on undercut anchors are discussed. Robust load capacities and relatively small displacements determined for demanding load and crack cycling tests demonstrated the suitability of anchors qualified according to a state-of-the-art qualification guideline

Characterization of the aspects of osteoprogenitor cell interactions with physical tetracalcium phosphate anchorage on titanium implant surfaces

Energy Technology Data Exchange (ETDEWEB)

Ko, Chia-Ling [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, Feng Chia University, Taichung 40724, Taiwan (China); Dental Medical Devices and Materials Research Center, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Yuan; Liou, Cian-Hua [Alliance Global Technology Co., Ltd., Kaohsiung Medical Device Special Zone in Southern Taiwan Science Park, Kaohsiung 82151, Taiwan (China); Chen, Wen-Cheng, E-mail: wencchen@fcu.edu.tw [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, Feng Chia University, Taichung 40724, Taiwan (China)

2015-04-01

Well-designed implants are used not only to modify the geometry of the implant but also to change the chemical properties of its surfaces. The present study aims to assess the biofunctional effects of tetracalcium phosphate (TTCP) particles as a physical anchor on the implant surface derived through sandblasting. The characteristics of the surface, cell viability, and alkaline phosphatase (ALP) activity toward osteoprogenitor cells (D1) were obtained. D1 cells were cultured on a plain surface that underwent sandblasting and acid etching (SLA) (control SLA group) and on different SLA surfaces with different anchoring TTCP rates (new test groups, M and H). The mean anchoring rates were 57% (M) and 74% (H), and the anchored thickness was estimated to range from 12.6 μm to 18.3 μm. Compared with the control SLA surface on Ti substrate, the new test groups with different TTCP anchoring rates (M and H) failed to improve cell proliferation significantly but had a well-differentiated D1 cell phenotype that enhanced ALP expression in the early stage of cell cultures, specifically, at day 7. Results suggest that the SLA surface with anchored TTCP can accelerate progenitor bone cell mineralization. This study shows the potential clinical application of the constructed geometry in TTCP anchorage on Ti for dental implant surface modification. - Highlights: • TTCP (tetracalcium phosphate) as a physical anchorage on implant is characterized. • Theoretical values of anchored thickness and capping areas were estimated. • TTCP anchored by sandblasting can accelerate progenitor bone cell mineralization. • TTCP anchored on SLA (sandblasting and acid etching) surface is a promising method.

Characterization of the aspects of osteoprogenitor cell interactions with physical tetracalcium phosphate anchorage on titanium implant surfaces

International Nuclear Information System (INIS)

Ko, Chia-Ling; Chang, Ya-Yuan; Liou, Cian-Hua; Chen, Wen-Cheng

2015-01-01

Well-designed implants are used not only to modify the geometry of the implant but also to change the chemical properties of its surfaces. The present study aims to assess the biofunctional effects of tetracalcium phosphate (TTCP) particles as a physical anchor on the implant surface derived through sandblasting. The characteristics of the surface, cell viability, and alkaline phosphatase (ALP) activity toward osteoprogenitor cells (D1) were obtained. D1 cells were cultured on a plain surface that underwent sandblasting and acid etching (SLA) (control SLA group) and on different SLA surfaces with different anchoring TTCP rates (new test groups, M and H). The mean anchoring rates were 57% (M) and 74% (H), and the anchored thickness was estimated to range from 12.6 μm to 18.3 μm. Compared with the control SLA surface on Ti substrate, the new test groups with different TTCP anchoring rates (M and H) failed to improve cell proliferation significantly but had a well-differentiated D1 cell phenotype that enhanced ALP expression in the early stage of cell cultures, specifically, at day 7. Results suggest that the SLA surface with anchored TTCP can accelerate progenitor bone cell mineralization. This study shows the potential clinical application of the constructed geometry in TTCP anchorage on Ti for dental implant surface modification. - Highlights: • TTCP (tetracalcium phosphate) as a physical anchorage on implant is characterized. • Theoretical values of anchored thickness and capping areas were estimated. • TTCP anchored by sandblasting can accelerate progenitor bone cell mineralization. • TTCP anchored on SLA (sandblasting and acid etching) surface is a promising method

Constrained Active Learning for Anchor Link Prediction Across Multiple Heterogeneous Social Networks.

Science.gov (United States)

Zhu, Junxing; Zhang, Jiawei; Wu, Quanyuan; Jia, Yan; Zhou, Bin; Wei, Xiaokai; Yu, Philip S

2017-08-03

Nowadays, people are usually involved in multiple heterogeneous social networks simultaneously. Discovering the anchor links between the accounts owned by the same users across different social networks is crucial for many important inter-network applications, e.g., cross-network link transfer and cross-network recommendation. Many different supervised models have been proposed to predict anchor links so far, but they are effective only when the labeled anchor links are abundant. However, in real scenarios, such a requirement can hardly be met and most anchor links are unlabeled, since manually labeling the inter-network anchor links is quite costly and tedious. To overcome such a problem and utilize the numerous unlabeled anchor links in model building, in this paper, we introduce the active learning based anchor link prediction problem. Different from the traditional active learning problems, due to the one-to-one constraint on anchor links, if an unlabeled anchor link a = ( u , v ) is identified as positive (i.e., existing), all the other unlabeled anchor links incident to account u or account v will be negative (i.e., non-existing) automatically. Viewed in such a perspective, asking for the labels of potential positive anchor links in the unlabeled set will be rewarding in the active anchor link prediction problem. Various novel anchor link information gain measures are defined in this paper, based on which several constraint active anchor link prediction methods are introduced. Extensive experiments have been done on real-world social network datasets to compare the performance of these methods with state-of-art anchor link prediction methods. The experimental results show that the proposed Mean-entropy-based Constrained Active Learning (MC) method can outperform other methods with significant advantages.

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

Science.gov (United States)

Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

2017-11-15

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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.

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.

Link Anchors in Images: Is there Truth?

NARCIS (Netherlands)

Aly, Robin; McGuinness, Kevin; Kleppe, Martijn; Ordelman, Roeland J.F.; O'Connor, Noel; de Jong, Franciska M.G.

2012-01-01

While automatic linking in text collections is well understood, little is known about links in images. In this work, we investigate two aspects of anchors, the origin of a link, in images: 1) the requirements of users for such anchors, e.g. the things users would like more information on, and 2)

Surface charge effects in protein adsorption on nanodiamonds

Science.gov (United States)

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

2015-03-01

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

BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

Directory of Open Access Journals (Sweden)

Wenjun Zhu

2017-09-01

Full Text Available We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein.

Science.gov (United States)

Krut, Oleg; Wiegmann, Katja; Kashkar, Hamid; Yazdanpanah, Benjamin; Krönke, Martin

2006-05-12

Two genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3ddeltaIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN.

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.

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.

The Effect of Mini and Midi Anchor Tests on Test Equating

Science.gov (United States)

Arikan, Çigdem Akin

2018-01-01

The main purpose of this study is to compare the test forms to the midi anchor test and the mini anchor test performance based on item response theory. The research was conducted with using simulated data which were generated based on Rasch model. In order to equate two test forms the anchor item nonequivalent groups (internal anchor test) was…

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

Science.gov (United States)

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

2014-08-08

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

Use of the ROC anchor in foot and ankle surgery. A retrospective study.

Science.gov (United States)

Kuwada, G T

1999-05-01

A retrospective study was conducted on the use of the ROC (Radial Osteo Compression) soft-tissue anchor in foot and ankle surgery. This article describes how the anchor is deployed, problematic aspects of using the anchor, and complications and success rates associated with the anchor in ankle stabilizations, posterior tibial tendon reconstruction, peroneus brevis tendon reconstruction after fracture of the base of the fifth metatarsal, and detachment and reattachment of the Achilles tendon. The ROC anchor consists of the anchor with nonabsorbable suture attached to the shaft, the deployment handle, and drill bits. The anchor and shaft are snapped into the deployment handle and inserted into the drill hole. Compression of the trigger deploys the anchor into the hole. The ROC anchor was found to be reliable, useful, and relatively easy to deploy, with outcomes similar to those of other soft-tissue anchors.

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)

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.

On the Robustness of Anchoring Effects in WTP and WTA Experiments

OpenAIRE

Drew Fudenberg; David K Levine; Zacharias Maniadis

2010-01-01

We reexamine the effects of the anchoring manipulation of Ariely, Loewenstein, and Prelec (2003) on the evaluation of common market goods and find very weak anchoring effects. We perform the same manipulation on the evaluation of binary lotteries, and find no anchoring effects at all. This suggests limits on the robustness of anchoring effects. (JEL C91, D12, D44)

Career Paths, Images and Anchors: A Study with Brazilian Professionals

Science.gov (United States)

Kilimnik, Zelia Miranda; de Oliveira, Luiz Claudio Vieira; Sant'anna, Anderson De Souza; Barros, Delba Teixeira Rodrigues

2011-01-01

This article analyses career anchors changes associated to images and professionals trajectories. Its main question: Do anchors careers change through time? We conducted twelve interviews involving professionals from the Administration Area, applying Schein's Career Anchors Inventory (1993). We did the same two years later. In both of them, the…

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

Stone anchors of India: Findings, classification and significance.

Digital Repository Service at National Institute of Oceanography (India)

Tripati, S.

Various types of stone anchors have been observed during inshore and offshore explorations along the east and west coasts of India. The earliest stone anchors of India have been recorded from the Harappan sites (3rd millennium BC), but their shape...

Investigation of suction anchor pullout capacity under undrained conditions

OpenAIRE

Jarand, Pollestad

2015-01-01

Master's thesis in Offshore technology Floating units are dependent on reliable mooring systems to ensure safety during marine operations. Suction anchors have proved to be a technologically viable and cost-effective concept. They are capable of precision installation, re-use, and provide large resistive capacity. This thesis investigates load capacity and failure modes of suction anchors subjected to vertical, horizontal (lateral), and incline loading. Suction anchor design co...

Pyramidal anchor stone from Baga waters of Goa, west coast of India

Digital Repository Service at National Institute of Oceanography (India)

Tripati, S.

. Pyramidal anchor stones have an apex hole which goes up to the round hole, however Goa anchor stone has no such perforation, but, instead has a rectangular cutting on the apex. The anchor stone is compared with Greek pyramidal anchor stones, and probably...

Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

International Nuclear Information System (INIS)

Haas, R.; Marshall, T.L.; Rosenberry, T.L.

1988-01-01

The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [ 125 ]I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine

Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning

Directory of Open Access Journals (Sweden)

Niek S. A. Crone

2018-01-01

Full Text Available A minimal model system for membrane fusion, comprising two complementary peptides dubbed “E” and “K” joined to a cholesterol anchor via a polyethyleneglycol spacer, has previously been developed in our group. This system promotes the fusion of large unilamellar vesicles and facilitates liposome-cell fusion both in vitro and in vivo. Whilst several aspects of the system have previously been investigated to provide an insight as to how fusion is facilitated, anchor positioning has not yet been considered. In this study, the effects of placing the anchor at either the N-terminus or in the center of the peptide are investigated using a combination of circular dichroism spectroscopy, dynamic light scattering, and fluorescence assays. It was discovered that anchoring the “K” peptide in the center of the sequence had no effect on its structure, its ability to interact with membranes, or its ability to promote fusion, whereas anchoring the ‘E’ peptide in the middle of the sequence dramatically decreases fusion efficiency. We postulate that anchoring the ‘E’ peptide in the middle of the sequence disrupts its ability to form homodimers with peptides on the same membrane, leading to aggregation and content leakage.

Testing methods of steel wi re ropes at the anchor

Directory of Open Access Journals (Sweden)

Stanislav Kropuch

2012-12-01

Full Text Available The present paper introduces an application of the acoustic andthermographic method in the defectoscopic testing of immobilesteel wire ropes at the most critical point, the anchor. Firstmeasurements and their results by these new defectoscopic methodsare shown. In defectoscopic tests at the anchor, the widelyused magnetic method gives unreliable results, and therefore presentsa problem for steel wire defectoscopy. Application of the two new methods in the steel wire defectoscopy at the anchor point will enableincreased safety measures at the anchor of steel wire ropes in bridge, roof, tower and aerial cable lift constructions.

LocateP: Genome-scale subcellular-location predictor for bacterial proteins

Directory of Open Access Journals (Sweden)

Zhou Miaomiao

2008-03-01

Full Text Available Abstract Background In the past decades, various protein subcellular-location (SCL predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP. Results LocateP combines many of the existing high-precision SCL identifiers with our own newly developed identifiers for specific SCLs. The LocateP pipeline was designed such that it mimics protein targeting and secretion processes. It distinguishes 7 different SCLs within Gram-positive bacteria: intracellular, multi-transmembrane, N-terminally membrane anchored, C-terminally membrane anchored, lipid-anchored, LPxTG-type cell-wall anchored, and secreted/released proteins. Moreover, it distinguishes pathways for Sec- or Tat-dependent secretion and alternative secretion of bacteriocin-like proteins. The pipeline was tested on data sets extracted from literature, including experimental proteomics studies. The tests showed that LocateP performs as well as, or even slightly better than other SCL predictors for some locations and outperforms

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.

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.

Synergism Analysis of Bedding Slope with Piles and Anchor Cable Support under Sine Wave Vehicle Load

Directory of Open Access Journals (Sweden)

Li Dan-Feng

2016-01-01

Full Text Available Slope instability under dynamic load is the technical difficulty in the engineering; the evaluation of slope stability under dynamic load and the control of dynamic load is particularly important. In this paper, taking the right side slope of K27+140 m~380 m typical section (K27 slope for short in Chongqing Fuling-Fengdu-Shizhu expresses highway as an example to calculate and analyze. The K27 slope is under sinusoidal vehicle load and supported by anchor cable and antislide pile to resist downslide strength; at the same time, the combined effect of them is studied. Three-dimensional finite element methodology (FEM is used to simulate the bedding slope with piles and anchor cable support; furthermore, the eigenvalue can be obtained. In order to reduce error of the elastic boundary conditions caused by the reflection effect of wavelengths, the combination of Lysmer surface viscous boundary and traditional ground support boundaries is utilized to analyze and calculate the time-histories during bedding slope under dynamic load. The dynamic response of pile anchor support to resist sliding force is obtained. The concept of the pile anchor supporting coordinate interval is put forward. Furthermore, it is verified that the pile anchor supporting coordinate interval can be used to evaluate the stability of the slope under dynamic load and provide a new method for the control of the dynamic load.

Assessing tether anchor labeling and usability in pickup trucks.

Science.gov (United States)

Klinich, Kathleen D; Manary, Miriam A; Malik, Laura A; Flannagan, Carol A; Jermakian, Jessica S

2018-04-03

The objective of this study was to investigate vehicle factors associated with child restraint tether use and misuse in pickup trucks and evaluate 4 labeling interventions designed to educate consumers on proper tether use. Volunteer testing was performed with 24 subjects and 4 different pickup trucks. Each subject performed 8 child restraint installations among the 4 pickups using 2 forward-facing restraints: a Britax Marathon G4.1 and an Evenflo Triumph. Vehicles were selected to represent 4 different implementations of tether anchors among pickups: plastic loop routers (Chevrolet Silverado), webbing routers (Ram), back wall anchors (Nissan Frontier), and webbing routers plus metal anchors (Toyota Tundra). Interventions included a diagram label, Quick Response (QR) Code linked to video instruction, coordinating text label, and contrasting text tag. Subjects used the child restraint tether in 93% of trials. However, tether use was completely correct in only 9% of trials. An installation was considered functional if the subject attached the tether to a tether anchor and had a tight installation (ignoring routing and head restraint position); 28% of subjects achieved a functional installation. The most common installation error was attaching the tether hook to the anchor/router directly behind the child restraint (near the top of the seatback) rather than placing the tether through the router and attaching it to the anchor in the adjacent seating position. The Nissan Frontier, with the anchor located on the back wall of the cab, had the highest rate of correct installations but also had the highest rate of attaching the tether to components other than the tether anchor (seat adjustor, child restraint storage hook, around head restraint). None of the labeling interventions had a significant effect on correct installation; not a single subject scanned the QR Code to access the video instruction. Subjects with the most successful installations spent extensive time

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.

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.

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

Ru-bis(pyridine)pyrazolate (bpp)-Based Water-Oxidation Catalysts Anchored on TiO2: The Importance of the Nature and Position of the Anchoring Group.

Science.gov (United States)

Francàs, Laia; Richmond, Craig; Garrido-Barros, Pablo; Planas, Nora; Roeser, Stephan; Benet-Buchholz, Jordi; Escriche, Lluís; Sala, Xavier; Llobet, Antoni

2016-04-04

Three distinct functionalisation strategies have been applied to the in,in-[{Ru(II)(trpy)}2(μ-bpp)(H2O)2](3+) (trpy=2,2':6',2''-terpyridine, bpp=bis(pyridine)pyrazolate) water-oxidation catalyst framework to form new derivatives that can adsorb onto titania substrates. Modifications included the addition of sulfonate, carboxylate, and phosphonate anchoring groups to the terpyridine and bis(pyridyl)pyrazolate ligands. The complexes were characterised in solution by using 1D NMR, 2D NMR, and UV/Vis spectroscopic analysis and electrochemical techniques. The complexes were then anchored on TiO2-coated fluorinated tin oxide (FTO) films, and the reactivity of these new materials as water-oxidation catalysts was tested electrochemically through controlled-potential electrolysis (CPE) with oxygen evolution detected by headspace analysis with a Clark electrode. The results obtained highlight the importance of the catalyst orientation with respect to the titania surface in regard to its capacity to catalytically oxidize water to dioxygen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Curcumin Protects Neurons from Glutamate-Induced Excitotoxicity by Membrane Anchored AKAP79-PKA Interaction Network

Directory of Open Access Journals (Sweden)

Kui Chen

2015-01-01

Full Text Available Now stimulation of AMPA receptor as well as its downstream pathways is considered as potential central mediators in antidepressant mechanisms. As a signal integrator which binds to AMPA receptor, A-kinase anchoring protein 79-(AKAP79- PKA complex is regarded as a potential drug target to exert neuroprotective effects. A well-tolerated and multitarget drug curcumin has been confirmed to exert antidepressant-like effects. To explore whether AKAP79-PKA complex is involved in curcumin-mediated antiexcitotoxicity, we detected calcium signaling, subcellular location of AKAP79-PKA complex, phosphorylation of glutamate receptor, and ERK and AKT cascades. In this study, we found that curcumin protected neurons from glutamate insult by reducing Ca2+ influx and blocking the translocation of AKAP79 from cytomembrane to cytoplasm. In parallel, curcumin enhanced the phosphorylation of AMPA receptor and its downstream pathways in PKA-dependent manner. If we pretreated cells with PKA anchoring inhibitor Ht31 to disassociate PKA from AKAP79, no neuroprotective effects were observed. In conclusion, our results show that AKAP79-anchored PKA facilitated the signal relay from AMPA receptor to AKT and ERK cascades, which may be crucial for curcumin-mediated antiexcitotoxicity.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

Directory of Open Access Journals (Sweden)

Wieczorek Andrew S

2010-09-01

Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

Archaeometallurgical investigation of the iron anchor from the Tantura F shipwreck

Energy Technology Data Exchange (ETDEWEB)

Aronson, A. [Faculty of Engineering, Tel Aviv University, Ramat Aviv 69978 (Israel); Ashkenazi, D., E-mail: dana@eng.tau.ac.il [Faculty of Engineering, Tel Aviv University, Ramat Aviv 69978 (Israel); Barkai, O.; Kahanov, Y. [Leon Recanati Institute for Maritime Studies, University of Haifa, Haifa 31905 (Israel)

2013-04-15

The Tantura F shipwreck was a coaster or a fishing vessel about 15.7 m long, discovered in the Dor/Tantura lagoon, Israel in 1995. It was dated to between the mid-7th and the end of the 8th centuries CE. Among the finds excavated were two T-shaped type iron anchors. Of the two anchors, one (anchor A) was thoroughly studied by archaeometallurgical methods in order to identify forge-welding lines, to determine the welding quality and to understand the manufacturing technology. The examinations included X-ray radiography, XRF analysis, optical microscopy, SEM/EDS observation and analysis, OES analysis and microhardness tests. The investigation included characterization of the composition, microstructure, thermal treatments, forge-welding junctions and slag analysis. The results revealed a heterogeneous microstructure, rich in glassy, fayalite and wüstite slag. Iron based phases included ferrite, pearlite, cementite and Widmanstätten plates, all typical to wrought iron. The forge-welds of Anchor A were located. Each arm was made of one piece, weighing about 2.5–3 kg and the shank was made of a few 1.5–2 kg pieces. The second anchor (anchor B) was only briefly examined visually and with a few radiographs, which support the results from anchor A. The research results revealed significant information about T-shaped anchors and their manufacturing process, including hot-working processes without any additional heat treatments, and folding techniques. The microstructure was similar to other ancient simple tools such as saws, sickles, axes and mortise chisels, and though the technology to make complicated structures and objects, such as swords, existed at that time, the anchors did not require this sophistication; thus simpler techniques were used, presumably because they were more cost-effective. - Highlights: ► Tantura F was a coaster dated to mid-7th–end-8th centuries. ► Two iron anchors were discovered at the Tantura F shipwreck-site. ► Anchor A was

Do budget balance rules anchor budget balance expectations? -- Some international evidence

OpenAIRE

Rülke, Jan-Christoph; Frenkel, Michael; Lis, Eliza

2013-01-01

This is the first study that analyzes whether budget balance expectations are anchored and whether budget balance rules effectively anchor expectations. To this end, we use a unique data set which covers budget balance expectations in 17 countries that implemented a budget balance rules. While our results are mixed concerning the general impact of budget balance rules on anchoring expectations, we do find that specific features of budget balance rules are important to successfully anchor budg...

Ultimate load capacities of expansion anchor bolts

International Nuclear Information System (INIS)

Czarnecki, R.M.; Manrique, M.A.; Samaddar, S.K.

1993-01-01

A summary of available experimental expansion anchor bolt test data is presented. These data were collected as part of programs by the nuclear industry to address generic issues related to verification of seismic adequacy of equipment in nuclear power plants. Some of the data presented are suitable for use in seismic probabilistic risk assessments. For example, mean values of ultimate strength, along with their standard deviation and coefficients of variation, for a range of most typical expansion anchor bolt sizes are presented. Effects of interaction between shear and tension, edge distance, spacing, and cracking of the concrete are presented in a manner that is more suitable for use in deterministic evaluations. Related industry programs to derive anchor bolt capacities are briefly discussed. Recommendations for areas of further investigation are also presented

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.

Biomimetic surface modification of polypropylene by surface chain transfer reaction based on mussel-inspired adhesion technology and thiol chemistry

Energy Technology Data Exchange (ETDEWEB)

Niu, Zhijun; Zhao, Yang; Sun, Wei; Shi, Suqing, E-mail: shisq@nwu.edu.cn; Gong, Yongkuan

2016-11-15

Highlights: • Biomimetic surface modification of PP was successfully conducted by integrating mussel-inspired technology, thiol chemistry and cell outer membranes-like structures. • The resultant biomimetic surface exhibits good interface and surface stability. • The obvious suppression of protein adsorption and platelet adhesion is also achieved. • The residue thoil groups on the surface could be further functionalized. - Abstract: Biomimetic surface modification of polypropylene (PP) is conducted by surface chain transfer reaction based on the mussel-inspired versatile adhesion technology and thiol chemistry, using 2-methacryloyloxyethylphosphorylcholine (MPC) as a hydrophilic monomer mimicking the cell outer membrane structure and 2,2-azobisisobutyronitrile (AIBN) as initiator in ethanol. A layer of polydopamine (PDA) is firstly deposited onto PP surface, which not only offers good interfacial adhesion with PP, but also supplies secondary reaction sites (-NH{sub 2}) to covalently anchor thiol groups onto PP surface. Then the radical chain transfer to surface-bonded thiol groups and surface re-initiated polymerization of MPC lead to the formation of a thin layer of polymer brush (PMPC) with cell outer membrane mimetic structure on PP surface. X-ray photoelectron spectrophotometer (XPS), atomic force microscopy (AFM) and water contact angle measurements are used to characterize the PP surfaces before and after modification. The protein adsorption and platelet adhesion experiments are also employed to evaluate the interactions of PP surface with biomolecules. The results show that PMPC is successfully grafted onto PP surface. In comparison with bare PP, the resultant PP-PMPC surface exhibits greatly improved protein and platelet resistance performance, which is the contribution of both increased surface hydrophilicity and zwitterionic structure. More importantly, the residue thiol groups on PP-PMPC surface create a new pathway to further functionalize such

Biomimetic surface modification of polypropylene by surface chain transfer reaction based on mussel-inspired adhesion technology and thiol chemistry

International Nuclear Information System (INIS)

Niu, Zhijun; Zhao, Yang; Sun, Wei; Shi, Suqing; Gong, Yongkuan

2016-01-01

Highlights: • Biomimetic surface modification of PP was successfully conducted by integrating mussel-inspired technology, thiol chemistry and cell outer membranes-like structures. • The resultant biomimetic surface exhibits good interface and surface stability. • The obvious suppression of protein adsorption and platelet adhesion is also achieved. • The residue thoil groups on the surface could be further functionalized. - Abstract: Biomimetic surface modification of polypropylene (PP) is conducted by surface chain transfer reaction based on the mussel-inspired versatile adhesion technology and thiol chemistry, using 2-methacryloyloxyethylphosphorylcholine (MPC) as a hydrophilic monomer mimicking the cell outer membrane structure and 2,2-azobisisobutyronitrile (AIBN) as initiator in ethanol. A layer of polydopamine (PDA) is firstly deposited onto PP surface, which not only offers good interfacial adhesion with PP, but also supplies secondary reaction sites (-NH 2 ) to covalently anchor thiol groups onto PP surface. Then the radical chain transfer to surface-bonded thiol groups and surface re-initiated polymerization of MPC lead to the formation of a thin layer of polymer brush (PMPC) with cell outer membrane mimetic structure on PP surface. X-ray photoelectron spectrophotometer (XPS), atomic force microscopy (AFM) and water contact angle measurements are used to characterize the PP surfaces before and after modification. The protein adsorption and platelet adhesion experiments are also employed to evaluate the interactions of PP surface with biomolecules. The results show that PMPC is successfully grafted onto PP surface. In comparison with bare PP, the resultant PP-PMPC surface exhibits greatly improved protein and platelet resistance performance, which is the contribution of both increased surface hydrophilicity and zwitterionic structure. More importantly, the residue thiol groups on PP-PMPC surface create a new pathway to further functionalize such

Do anchor investors create value for initial public offerings? An empirical investigation

Directory of Open Access Journals (Sweden)

Seshadev Sahoo

2017-12-01

Full Text Available The concept of anchor investors was introduced by the market regulator, Securities Exchange Board of India (SEBI, to bring transparency in the book building mechanism. We examine anchor investors' investment in initial public offerings (IPOs to determine how they create value for issuing firms and participating investors. Using a database of 135 IPOs issued in the Indian market through book building mechanism during 2009–2014, we find that anchor investors' investment in IPOs reduces underpricing. Larger subscription from retail investors for anchor-supported IPOs indicates that anchor investors' participation is viewed as a credible attestation of quality of the issue. We document that anchor-supported IPOs are more liquid and less volatile in the short run. We also find that by controlling for other factors such as offer size, subscription rate and age of the firm, a part of the underpricing is reduced by anchor investors.

Considerations on the design of through-wall anchors

International Nuclear Information System (INIS)

Ricklefs, Ulf

2012-01-01

Connections to existing buildings are often the most difficult planning challenge for the realization of construction measures in case of piping system replacements in nuclear power plants. This is due to restricted space or limited load reserves of the building structure. Usually the realization of support connections to the existing buildings is achieved by anchor bolts. But in critical cases the preferred alternative solution uses through-wall anchors. Up to now uniform assessment thresholds are not available, no technical guidelines or regulations for construction variants exist. Through-wall anchors allow significantly higher load capacities for tensile and shear loads but require enhanced planning and realization efforts.

Anchor enhanced capsulorraphy in bunionectomies using an L-shaped capsulotomy.

Science.gov (United States)

Gould, John S; Ali, Sheriff; Fowler, Rachel; Fleisig, Glenn S

2003-01-01

The objective of this study was to investigate potential benefit of a suture anchor-enhanced capsulorraphy in the early maintenance of correction in bunionectomies. We compared, retrospectively, in successive series, the loss of correction of the Hallux Valgus (HV) and intermetatarsal (IM) angle, in those repaired with an L-shaped capsulorraphy enhanced with anchors to those without. Intraoperative and second week postoperative simulated weightbearing anterior posterior (AP) X-rays were used to evaluate results. By using only intraoperative and early postoperative X-rays, we should have effectively eliminated extraneous factors that might have influenced our results. A Total of 106 cases were investigated, 65 of which were repaired using anchors, the remaining 41 without. In the anchor group, 38 underwent a proximal metatarsal concentric shelf osteotomy (CSO)/modified McBride procedure, while the remaining 27 had a distal Chevron correction. In the without-anchor group, 21 had a CSO/modified McBride procedure while 20 underwent the Chevron procedure. In the without-anchor group, the average HV and IM loss of correction was 4.60 degrees (range, -2 to 21 degrees) and 0.6 degrees (range, -1 to 9 degrees) respectively. In the anchor group, the corresponding loss was 2.8 degrees (range, -3 to 17 degrees) and 0.6 degrees (range, -2 to 14 degrees) respectively. These results, when statistically analyzed, demonstrated that while the IM angle change was not statistically significant, the HV angle change was statistically significant, implying that the anchor plays a significant role in maintaining the surgical correction in both the distal Chevron and CSO/ modified McBride bunionectomies.

Stone anchors from the Okhamandal region, Gujarat Coast, India

Digital Repository Service at National Institute of Oceanography (India)

Sundaresh; Gaur, A.S.; Gudigar, P.; Tripati, S.; Vora, K.H.; Bandodkar, S.N.

During marine archaeological explorations since 1983, off Dwarka, a large number of stone anchors were discovered and dated to 1400 BC, comparing with anchors found in Mediterranean waters. In recent archaeological explorations off Dwarka, Bet...

Spatial distribution of proteins in the quagga mussel adhesive apparatus.

Science.gov (United States)

Rees, David J; Hanifi, Arash; Manion, Joseph; Gantayet, Arpita; Sone, Eli D

2016-01-01

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

Preclinical trial of a novel surface architecture for improved primary fixation of cementless orthopaedic implants.

Science.gov (United States)

Harrison, Noel; Field, John R; Quondamatteo, Fabio; Curtin, William; McHugh, Peter E; Mc Donnell, Pat

2014-09-01

A new surface architecture for cementless orthopaedic implants (OsteoAnchor), which incorporates a multitude of tiny anchor features for enhancing primary fixation, was tested in an ovine hemi-arthroplasty pilot study. Test animals were implanted with a hip stem component incorporating the OsteoAnchor surface architecture produced using additive layer manufacturing and control animals were implanted with stems containing a standard plasma sprayed titanium coating. Intra-operative surgeon feedback indicated that superior primary fixation was achieved for the OsteoAnchor stems and rapid return to normal gait and load bearing was observed post-operation. Following a 16-week recovery time, histological evaluation of the excised femurs revealed in-growth of healthy bone into the porous structure of the OsteoAnchor stems. Bone in-growth was not achieved for the plasma sprayed stems. These results indicate the potential for the OsteoAnchor surface architecture to enhance both the initial stability and long term lifetime of cementless orthopaedic implants. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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

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

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.

Anchoring platinum on graphene using metallic adatoms: a first principles investigation

International Nuclear Information System (INIS)

Sen, F G; Alpas, A T; Qi, Y

2012-01-01

First principles calculations based on spin-polarized density functional theory were used to identify metallic adatoms that would strengthen the Pt(111)/graphene interface (with a low work of separation of 0.009 J m -2 ), when the adatom was placed between the Pt(111) and the graphene. It was shown that the strength of the Pt-adatom bond, which had a metallic character, increased with the amount of charge transferred from the adatom to the Pt. The carbon-adatom bond, on the other hand, had a mixed ionic and covalent character and was weaker than the Pt-adatom bond for each of the 25 elements considered. Consequently, the total Pt(111)/graphene interface strength and, hence, the anchoring effect of the adatom were controlled by the carbon-adatom bond strength. Metals with unfilled d orbitals increased the Pt/graphene interface strength to above 0.5 J m -2 . The carbon-adatom bond strength was proportional to the ratio between the charge transferred from the adatom to the graphene (ΔZ C ) and the charge transferred to the Pt surface (ΔZ Pt ); i.e., the ΔZ C /ΔZ Pt ratio defined the ability of an adatom to anchor Pt to graphene. For Ir, Os, Ru, Rh and Re, ΔZ C /ΔZ Pt > 1.0, making these elements the most effective adatoms for anchoring Pt to graphene. (paper)

Characterization of Three Different Unusual S-Layer Proteins from Viridibacillus arvi JG-B58 That Exhibits Two Super-Imposed S-Layer Proteins

Science.gov (United States)

Günther, Tobias J.; Raff, Johannes; Pollmann, Katrin

2016-01-01

Genomic analyses of Viridibacillus arvi JG-B58 that was previously isolated from heavy metal contaminated environment identified three different putative surface layer (S-layer) protein genes namely slp1, slp2, and slp3. All three genes are expressed during cultivation. At least two of the V. arvi JG-B58 S-layer proteins were visualized on the surface of living cells via atomic force microscopy (AFM). These S-layer proteins form a double layer with p4 symmetry. The S-layer proteins were isolated from the cells using two different methods. Purified S-layer proteins were recrystallized on SiO2 substrates in order to study the structure of the arrays and self-assembling properties. The primary structure of all examined S-layer proteins lack some features that are typical for Bacillus or Lysinibacillus S-layers. For example, they possess no SLH domains that are usually responsible for the anchoring of the proteins to the cell wall. Further, the pI values are relatively high ranging from 7.84 to 9.25 for the matured proteins. Such features are typical for S-layer proteins of Lactobacillus species although sequence comparisons indicate a close relationship to S-layer proteins of Lysinibacillus and Bacillus strains. In comparison to the numerous descriptions of S-layers, there are only a few studies reporting the concomitant existence of two different S-layer proteins on cell surfaces. Together with the genomic data, this is the first description of a novel type of S-layer proteins showing features of Lactobacillus as well as of Bacillus-type S-layer proteins and the first study of the cell envelope of Viridibacillus arvi. PMID:27285458

Design Analysis and Observed Performance of a Tieback Anchored Pile Wall in Sand

Directory of Open Access Journals (Sweden)

Jian-Yong Han

2017-01-01

Full Text Available This paper aims to study the design process and service performance of a deep excavation supported by tieback anchored pile walls. The design procedure and design approaches for deep excavation in China are described. Based on the excavation case history for Shenyang, China, design results obtained using the elastic method and the finite element method (FEM are compared and analyzed. Special emphasis is given to the analysis of horizontal wall deformations, internal forces in the wall, earth pressures on the wall, ground surface settlements, and stabilities of the excavation. The similarities and differences between the Chinese code (JGJ 120-2012 and the European code (EN 1997-1 for the design of geotechnical structures are presented based on a design example. Through the comparison, it is indicated that the Chinese code focuses on the design result, while the European code focuses on the design process. The crucial construction methods for reducing construction risk based on the excavation case history are described. The mechanical behaviors of the excavation retained by an anchored pile wall were investigated by analyzing observed field cases. The results provide good, practical guidelines for the design and construction of a tieback anchored pile wall retained excavation in sandy soil.

Effectiveness of TAD-anchored maxillary protraction in late mixed dentition.

Science.gov (United States)

Feng, Xiaoxia; Li, Jianhua; Li, Yu; Zhao, Zhihe; Zhao, Sen; Wang, Jue

2012-11-01

To evaluate the effectiveness of temporary anchorage device (TAD)-anchored maxillary protraction (MP) in terms of the skeletal and dentoalveolar changes and to compare it with traditional tooth-anchored MP. A computerized literature search for relative randomized controlled trials and prospective controlled trials was performed in PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Embase, CNKI, and Google Scholar, complemented with manual search. Data extraction and quality assessment were carried out by two reviewers independently. Meta-analysis was followed when possible; otherwise, description was done. Forty articles were found, among which four trials were qualified for meta-analysis. The results showed that there was significant difference between TAD-anchored MP and untreated control in terms of maxillary advancement (weighted mean differences (WMD) 3.08 mm; 95% CI: 1.61 to approximately 4.56; P TAD-anchored MP might have a greater maxillary advancement effect and might reduce skeletal and dental side effects, compared with tooth-anchored MP.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

Science.gov (United States)

Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

2017-01-15

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this

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...

Empirical evidence for resource-rational anchoring and adjustment.

Science.gov (United States)

Lieder, Falk; Griffiths, Thomas L; M Huys, Quentin J; Goodman, Noah D

2018-04-01

People's estimates of numerical quantities are systematically biased towards their initial guess. This anchoring bias is usually interpreted as sign of human irrationality, but it has recently been suggested that the anchoring bias instead results from people's rational use of their finite time and limited cognitive resources. If this were true, then adjustment should decrease with the relative cost of time. To test this hypothesis, we designed a new numerical estimation paradigm that controls people's knowledge and varies the cost of time and error independently while allowing people to invest as much or as little time and effort into refining their estimate as they wish. Two experiments confirmed the prediction that adjustment decreases with time cost but increases with error cost regardless of whether the anchor was self-generated or provided. These results support the hypothesis that people rationally adapt their number of adjustments to achieve a near-optimal speed-accuracy tradeoff. This suggests that the anchoring bias might be a signature of the rational use of finite time and limited cognitive resources rather than a sign of human irrationality.

Thermal anchoring of wires in large scale superconducting coil test experiment

International Nuclear Information System (INIS)

Patel, Dipak; Sharma, A.N.; Prasad, Upendra; Khristi, Yohan; Varmora, Pankaj; Doshi, Kalpesh; Pradhan, S.

2013-01-01

Highlights: • We addressed how thermal anchoring in large scale coil test is different compare to small cryogenic apparatus? • We did precise estimation of thermal anchoring length at 77 K and 4.2 K heat sink in large scale superconducting coil test experiment. • We addressed, the quality of anchoring without covering entire wires using Kapton/Teflon tape. • We obtained excellent results in temperature measurement without using GE Varnish by doubling estimated anchoring length. -- Abstract: Effective and precise thermal anchoring of wires in cryogenic experiment is mandatory to measure temperature in milikelvin accuracy and to avoid unnecessary cooling power due to additional heat conduction from room temperature (RT) to operating temperature (OT) through potential, field, displacement and stress measurement instrumentation wires. Instrumentation wires used in large scale superconducting coil test experiments are different compare to cryogenic apparatus in terms of unique construction and overall diameter/area due to errorless measurement in large time-varying magnetic field compare to small cryogenic apparatus, often shielded wires are used. Hence, along with other variables, anchoring techniques and required thermal anchoring length are entirely different in this experiment compare to cryogenic apparatus. In present paper, estimation of thermal anchoring length of five different types of instrumentation wires used in coils test campaign at Institute for Plasma Research (IPR), India has been discussed and some temperature measurement results of coils test campaign have been presented

Results of the ANCHOR prospective, multicenter registry of EndoAnchors for type Ia endoleaks and endograft migration in patients with challenging anatomy.

Science.gov (United States)

Jordan, William D; Mehta, Manish; Varnagy, David; Moore, William M; Arko, Frank R; Joye, James; Ouriel, Kenneth; de Vries, Jean-Paul

2014-10-01

Proximal attachment site complications continue to occur after endovascular repair of abdominal aortic aneurysms (EVAR), specifically type Ia endoleak and endograft migration. EndoAnchors (Aptus Endosystems, Sunnyvale, Calif) were designed to enhance endograft proximal fixation and sealing, and the current study was undertaken to evaluate the potential benefit of this treatment. During the 23-month period ending in December 2013, 319 subjects were enrolled at 43 sites in the United States and Europe. EndoAnchors were implanted in 242 patients (75.9%) at the time of an initial EVAR procedure (primary arm) and in 77 patients with an existing endograft and proximal aortic neck complications (revision arm). Technical success was defined as deployment of the desired number of EndoAnchors, adequate penetration of the vessel wall, and absence of EndoAnchor fracture. Procedural success was defined as technical success without a type Ia endoleak at completion angiography. Values are expressed as mean ± standard deviation and interquartile range. The 238 male (74.6%) and 81 female (25.4%) subjects had a mean age of 74.1 ± 8.2 years. Aneurysms averaged 58 ± 13 (51-63) mm in diameter at the time of EndoAnchor implantation (core laboratory measurements). The proximal aortic neck averaged 16 ± 13 (7-23) mm in length (42.7% <10 mm and 42.7% conical) and 27 ± 4 mm (25-30 mm) in diameter; infrarenal neck angulation was 24 ± 15 (13-34) degrees. The number of EndoAnchors deployed was 5.8 ± 2.1 (4-7). Technical success was achieved in 303 patients (95.0%) and procedural success in 279 patients (87.5%), 217 of 240 (89.7%) and 62 of 77 (80.5%) in the primary and revision arms, respectively. There were 29 residual type Ia endoleaks (9.1%) at the end of the procedure. During mean follow-up of 9.3 ± 4.7 months, 301 patients (94.4%) were free from secondary procedures. Among the 18 secondary procedures, eight were performed for residual type Ia endoleaks and the others

Note on the hydrodynamic description of thin nematic films: Strong anchoring model

KAUST Repository

Lin, Te-Sheng; Cummings, Linda J.; Archer, Andrew J.; Kondic, Lou; Thiele, Uwe

2013-01-01

We discuss the long-wave hydrodynamic model for a thin film of nematic liquid crystal in the limit of strong anchoring at the free surface and at the substrate. We rigorously clarify how the elastic energy enters the evolution equation for the film thickness in order to provide a solid basis for further investigation: several conflicting models exist in the literature that predict qualitatively different behaviour. We consolidate the various approaches and show that the long-wave model derived through an asymptotic expansion of the full nemato-hydrodynamic equations with consistent boundary conditions agrees with the model one obtains by employing a thermodynamically motivated gradient dynamics formulation based on an underlying free energy functional. As a result, we find that in the case of strong anchoring the elastic distortion energy is always stabilising. To support the discussion in the main part of the paper, an appendix gives the full derivation of the evolution equation for the film thickness via asymptotic expansion. © 2013 AIP Publishing LLC.

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....

GPI anchor biosynthesis in yeast : phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

NARCIS (Netherlands)

Canivenc-Gansel, E; Imhof, I; Reggiori, F; Burda, P; Conzelmann, A; Benachour, A; Reggiori, Fulvio

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant

BayesMotif: de novo protein sorting motif discovery from impure datasets.

Science.gov (United States)

Hu, Jianjun; Zhang, Fan

2010-01-18

Protein sorting is the process that newly synthesized proteins are transported to their target locations within or outside of the cell. This process is precisely regulated by protein sorting signals in different forms. A major category of sorting signals are amino acid sub-sequences usually located at the N-terminals or C-terminals of protein sequences. Genome-wide experimental identification of protein sorting signals is extremely time-consuming and costly. Effective computational algorithms for de novo discovery of protein sorting signals is needed to improve the understanding of protein sorting mechanisms. We formulated the protein sorting motif discovery problem as a classification problem and proposed a Bayesian classifier based algorithm (BayesMotif) for de novo identification of a common type of protein sorting motifs in which a highly conserved anchor is present along with a less conserved motif regions. A false positive removal procedure is developed to iteratively remove sequences that are unlikely to contain true motifs so that the algorithm can identify motifs from impure input sequences. Experiments on both implanted motif datasets and real-world datasets showed that the enhanced BayesMotif algorithm can identify anchored sorting motifs from pure or impure protein sequence dataset. It also shows that the false positive removal procedure can help to identify true motifs even when there is only 20% of the input sequences containing true motif instances. We proposed BayesMotif, a novel Bayesian classification based algorithm for de novo discovery of a special category of anchored protein sorting motifs from impure datasets. Compared to conventional motif discovery algorithms such as MEME, our algorithm can find less-conserved motifs with short highly conserved anchors. Our algorithm also has the advantage of easy incorporation of additional meta-sequence features such as hydrophobicity or charge of the motifs which may help to overcome the limitations of

Glypican-1 mediates both prion protein lipid raft association and disease isoform formation.

Directory of Open Access Journals (Sweden)

David R Taylor

2009-11-01

Full Text Available In prion diseases, the cellular form of the prion protein, PrP(C, undergoes a conformational conversion to the infectious isoform, PrP(Sc. PrP(C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs. We show that heparin displaces PrP(C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C. We then utilised a transmembrane-anchored form of PrP (PrP-TM, which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C from rafts, promoting its endocytosis. Glypican-1 and PrP(C colocalised on the cell surface and both PrP(C and PrP(Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C and PrP(Sc in lipid rafts.

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.

Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

Science.gov (United States)

Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

2014-01-01

Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

Energy Technology Data Exchange (ETDEWEB)

Kind, Barbara, E-mail: barbara.kind@uniklinikum-dresden.de [Children' s Hospital, Technical University Dresden, D-01307 Dresden (Germany); Koehler, Katrin, E-mail: katrin.koehler@uniklinikum-dresden.de [Children' s Hospital, Technical University Dresden, D-01307 Dresden (Germany); Lorenz, Mike, E-mail: mlorenz@mpi-cbg.de [Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden (Germany); Huebner, Angela, E-mail: angela.huebner@uniklinikum-dresden.de [Children' s Hospital, Technical University Dresden, D-01307 Dresden (Germany)

2009-12-11

The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

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.

Cyclic biomechanical testing of biocomposite lateral row knotless anchors in a human cadaveric model.

Science.gov (United States)

Barber, F Alan; Bava, Eric D; Spenciner, David B; Piccirillo, Justin

2013-06-01

The purpose of this study was to assess the mechanical performance of biocomposite knotless lateral row anchors based on both anchor design and the direction of pull. Two lateral row greater tuberosity insertion sites (anterior and posterior) were identified in matched pairs of fresh-frozen human cadaveric shoulders DEXA (dual energy X-ray absorptiometry) scanned to verify comparability. The humeri were stripped of all soft tissue and 3 different biocomposite knotless lateral row anchors: HEALIX Knotless BR (DePuy Mitek, Raynham MA), BioComposite PushLock (Arthrex, Naples, FL), and Bio-SwiveLock (Arthrex). Fifty-two anchors were distributed among the insertion locations and tested them with either an anatomic or axial pull. A fixed-gauge loop (15 mm) of 2 high-strength sutures from each anchor was created. After a 10-Nm preload, anchors were cycled from 10 to 45 Nm at 0.5 Hz for 200 cycles and tested to failure at 4.23 mm/second. The load to reach 3 mm and 5 mm displacement, ultimate failure load, displacement at ultimate failure, and failure mode were recorded. Threaded anchors (Bio-SwiveLock, P = .03; HEALIX Knotless, P = .014) showed less displacement with anatomic testing than did the nonthreaded anchor (BioComposite PushLock), and the HEALIX Knotless showed less overall displacement than did the other 2 anchors. The Bio-SwiveLock exhibited greater failure loads than did the other 2 anchors (P < .05). Comparison of axial and anatomic loading showed no maximum load differences for all anchors as a whole (P = .1084). Yet, anatomic pulling produced higher failure loads than did axial pulling for the Bio-SwiveLock but not for the BioComposite PushLock or the HEALIX Knotless. The nonthreaded anchor (BioComposite PushLock) displayed lower failure loads than did both threaded anchors with axial pulling. Threaded biocomposite anchors (HEALIX Knotless BR and Bio-SwiveLock) show less anatomic loading displacement and higher axial failure loads than do the nonthreaded

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.